NAVIGATE THIS ARTICLE: Steps >RNA Isolation, Probe Generation>Electrophores is, Transfer > Hybridization, DetectionNorthern analysis remains a standard method for det ection and quantitation of mRNA levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and nuclease protection assays.Northern analysis provides a direct relative compa rison of message abundance between samples on a single membran e. It is the preferred method for determining transcript size and for det ecting alternatively spliced transcripts . The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., integrity of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresi s in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally versatile in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and oligonucleotides can all be used as hybridization probes. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an intron) may be used as probes. Despite these advantages, there are limitations associated with Northern analysis. First, if RNA samples are even slightly deg raded, the quality ofthe data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. Second, a standard Northern procedure is, in general, less sensitive than nuclease protection assays and RT-PCR, although improvements in sensitivity can be achieved by using high specific activity antisense RNA probes, optimized hybridization buffers and positively charged nylon membranes. Sensitivity can be further improved with oligo dT selection for enrichment of mRNA, since physical constraints of gel electrophoresis and membrane transfer limit the amount of RNA that can be analyzed without loss of resolution and saturation ofthe transfer membrane. Ambion's NorthernMax™ reagents in combination with ULTRAhyb™ (see below) can dramatically increase the sensitivity ofNortherns to the level of nuclease protection assays. A third limitation ofNorthern blotting has been the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. This process can be time consuming and problematic, since harsh treatment is required to strip conventional probes from blots. Steps Involved in Northern Analysis
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Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisSubmitted by:-Rashid hussain
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisRoll number:- A55,Reg no:-
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisdescription. Often words are inadequate to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisBiotechnology at Lovely Professional
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisfriends for their blessings and
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis2.BACKGROUND INFORMATION-NORTHERN BLOT TECHNIQUE
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis6.ADVANTAGE OF NORTHERN BLOT
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis10.GROSS LINKING NYLON MEMBRANE NORTHERN BLOT
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis14.NORTHERN BLOT REFRENCE
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisHistory of NorthernBlotting
Northernblotting is an RNAblotting techniquewhich was
developedin 1977 by Alwineet al. at StanfordUniversity It was
namedaftertheSouthernblottechniquewhich blots for DNA,
inventedbyEdwin M. Southernin 1975.Westernblot, a methodfor
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisblotting for proteinsis also a play on the Southernblot namingand
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisNorthernanalysis despite its age in the high tech world of Real Time
PCR, nucleaseprotectionassays(RPAs) and microarrays, is still
the gold-standardfor the detectionand quantitationof mRNAlevels.
This is becausenorthernblot analysis allowsa direct comparisonof
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisthe messengerRNAabundancebetweensampleson a single
membrane.
In northernblot the main differencebetweenthe other blotting
techniquesis that RNAis the factor being detected. Also, due to the
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisfact that RNAis usually single-stranded,it createscomplex
secondarystructureswhich affect its migrationand hence
denaturingconditionsare used to run the gels (unlike Southern).
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisRNAis separatedout by RNAgel electrophoresis(usually agarose
gel electrophoresis), subsequenttransfer to membrane,
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysishybridization probes may be DNA or RNA in northern
blotting.
A variant of the procedure known as the reverse northern
blot was occasionally (although, infrequently) used. In this
procedure, the substrate nucleic acid (that is affixed to the
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysismembrane) is a collection of isolated DNA fragments, and
the probe is RNA extracted from a tissue and radioactively
labelled.
The use of DNA microarrays that have come into
widespread use in the late 1990s and early 2000s is more
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisakin to the reverse procedure, in that they involve the use of
isolated DNA fragments affixed to a substrate, and
hybridization with a probe made from cellular RNA. Thus
the reverse procedure, though originally uncommon,
enabled the one-at-a-time study of gene expression using
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisnorthern analysis to evolve into gene expression profiling,in
which many (possibly all) of the genes in an organism may
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis
Northern blots have been superceded in most areas by
Real Time PCR and microarray approaches. It is not often
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisThe northern blot protocol and its variations are used
however in molecular biology research to:
• a gold-standard for the direct study of gene expression at
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• study RNA degradation
• study RNA splicing - can detect alternatively spliced
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• study IRES (internal ribosomal entry site) - to remove
possibility of RNA digestion vs 2nd cistron translation.
• often used to confirm and check transgenic / knockout
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisDisadvantagesof NorthernBlotting
Often radioactivity is used. This preventsease of performing
it, use and disposal. New methodsof non-radioactivedetection
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysishave been generatedallowingnon-radioactivedetection. See
Pierce.
• The whole processof northernblotting takes a long time
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• If RNAsamplesare even slightly degradedby RNases, the
quality of the data and quantitationof expressionis quite
negativelyaffected.
• The standardnorthernblot methodis relatively less sensitive
than nucleaseprotectionassaysand RT-PCR. The sensitivity
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisof northernblots may be increasedwith the use of nylon
positively-chargedmembranes,use of a highly specific
antisenseprobe.
• Detectionwith multiple probesis a problem.Often, the
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisdetectionwith a secondprobe. This is a problemas harsh
conditionsare requiredto strip off probesfrom the blot and is
also time consuming.Also, there is a limit to the amountof
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisAdvantagesof NorthernBlotting
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• Often it is used as a confirmationor check
• Often a gold-standard
• it is a versatile protocol as it can allow the usageof many
typesof probes(vs Real time PCR) including:radiolabeled
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisand non-radiolabeled, in vitro transcribedRNAand even
oligonucleotidessuch as primers.
• Sequenceswith even partial homology,unlike real time PCR
or other methodscan be used as hybridizationprobes(i.e
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysissequencefrom different speciesfor homologyanalysis, or
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisNorthernBlot Protocol
As mentionedthe steps in northernblotting include:
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis3. Transfer to membrane(usually positively chargednylon as RNA
is negativelycharged)
4. Cross-linking of RNAto membrane(usually by UV-crosslinking
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis5. Hybridization
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• 20mMEDTA:20mLof 0.5M
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• pH to 7.0 after both are added
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis
* *Right before using add 1000ugdenaturedRNAsefree CT DNA
(heat to 95o for 3 min to denature) 5000ugyeast RNA
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• 52. 6 mls of formaldehyde
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• 7 mls 10x MOPS
• 58.38 mls H2O
• Put gel in solutionby heating. Let gel cool to 60°C, then add
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• Pour gel in fumehood if possible
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisAfter isolatingRNA, the RNAmust be separatedout on a gel
(usually agarose).
NorthernBlot Transfer to Nylon MembraneProtocol
After runningthe RNAgel, washthe gel with distilledwater put on posiblotter.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisLayersfromtop to bottomare:
• sponge
• 3-4 Whatmanpaperscut to size ( both of these up aboveshouldbe soakedin 10X
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• Note: Makesure the gel overlaysthe maskso that it can seal membranewith line of
the top of gel and the right top corner marked3 piecesslightly bigger3M paper
hard plastic with holes
Clampon and makesure there is a goodseal. Use 75-80 mmHg of pressurefor 1 hr or
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisBlot membranedry
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis• Washfor a couple of minutesin hot 2X SSPE/0.1%SDS
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis
Pre Hybridizationfor NorthernBlot
1. Pre Hybridizefor 6 hours-overnight
2. Set oven to 65°C heat HB buffer to put SDSin solution
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis3. Roll up membraneinside piece of meshand put into hybridizaton
tube(2XSSPE/0.1%SDS)
4. Checkfor air bubbles
5. Put tube in oven balancedwith anothertube on oppositeside
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisLabelingprobefor NorthernBlotting
1. Put 25ng of probein a total volumeof 11uL with ddH2O
2. Denature@ 95°C 3 min. This is to get the probesingle strandedand
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis3. Quick cool on wet ice. This is to keep the probesingle stranded,free of
secondarystructureand not allowreannealing(or reformingof
secondarystructures).
4. Add 4ul High Prime(BoehringerMannheim)5ul alpha radiolabeledP-32
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis5. Incubate10-15 min 37°C
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis8. Add 50ul sampleto columnand spin for 2.5 min in clinical centrifuge.
This is to purify probeaway fromlabel.
9. Throwaway column.
10.Mix in anew tube 100ugCT-DNA50ug Yeast RNAin a 0.5mLtube.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis11.Denature95°3 min
12.Add to hybrid tube mix, hybridizeat 65°C for 20-36 hrs
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisHybridizefor 36-48 hours then wash.
1. Heat up 2X SSPE/0.1%SDS (wash) heat up to 65°C (Use microwaveor
water bath to warmsolution)
2. Take out tube and pour liquid into hot radioactivewaste container.
3. Rinse with 10ml washdo this twice and pour waste out.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis4. Put in 40 to 50 mls of washand shakevigoursly.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis8. Pour out to waste container(sink if not hot or toxic) and makesure it is
no longer hot and then take out membrane.
9. Washon shaker with 0.2XSSPEw/ 0.1%SDS at RT for 15 min.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis11.Expose
Tips for NorthernBlot
If you currently have a preferredmethodfor isolatingRNA, continueto use it.
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern AnalysisAlwaysrun an agarosegel of your RNAto assessthe quality. Do not only rely
on spectrophotometryresults.
* Use DEPCtreatedwater and bakedglassware.This solution, absolutely,
positively must be Rnasefree. Rnaseis everywhere!Wear gloves, use only
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysisbakedglassware,or virgin plastic. DePCtreat your water, buffers (except
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis1. AlwineJC, KempDJ, Stark GR (1977). "Methodfor detectionof specific RNAsin
agarosegels by transfer to diazobenzyloxymethyl-paper and hybridizationwith
DNAprobes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350-4.
2. W. Neal Burnette(April 1981). "'Westernblotting': electrophoretictransferof
proteinsfromsodiumdodecyl sulfate — polyacrylamidegels to unmodified
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
strip conventional probes from blots.
Steps Involved in Northern Analysis3. http://www.molecularstation.com/rna/northern-blot/
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to
Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,
such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively
spliced transcripts.
The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the
RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel
under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as
hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might
contain an intron) may be used as probes.
Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of
the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target
molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern
procedure is, in general, less sensitive than nuclease protection assays and
RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical
constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination
with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of
Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip
the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to