Presentation 33333

8/2/2019 Presentation 33333

1/62

RNA ISOLATION and

PURIFICATION


2/62

SELECTING A PURIFICATION STRATEGYDo Your Experiments Require Total RNA or mRNA?

the researcher must be has to make when detecting or quantitating

RNA is whether to isolate total RNA or poly(A)-selected RNA(mRNA).

This choosing is complicated by:the bewildering array of RNA isolation kits available in the marketplace.

the downstream application influences this choice.

total RNA might suffice for most applications.

the starting material for applications ranging from the detection of anmRNA species by Northern hybridization to quantitation of a messageby RT_PCR.

You remember that:mRNA comprises (


3/62

How do resolve this problem?

If data generated with total RNA do not meet your expectations,

using poly(A) RNA instead might provide the( sensitivity)andspecificitythat your application requires.

You must be detect The pros and cons of each

application.

Two situations where using poly(A) RNA is essential are:1.cDNA library construction.

2.preparation of labeled cDNA for hybridization to genearrays.


4/62

Northern blotting or Northern hybridization

is a technique for detection of specific RNA sequences.

Northern blotting was developed by James Alwine and George Stark at Stanford

University and was named such by analogy to Southern blotting.

Northern blotting involves the following steps:

1) Total cellular RNA or mRNA is size-separated by denaturing agarose gel

electrophoresis,

2) 2) The separated RNA is transferred onto a nylon membrane;

3) 3) The RNA is then detected with isotopic or non-isotopic labeled complementary

DNA or RNA probe


5/62


6/62

Northern Hybridizations

Northern analysis is the only technique available that can determinethe molecular weight of an mRNA species.

Total RNA is most commonly used in this assay.

Since only very small amounts of poly(A) RNAare present,

make sure that it is feasible and practical to obtainenough starting cells or tissue.

Example .if you use 30micro gram of poly(A) RNA in a Northern ,which is theamount found in approximately 1mg of total RNA.

Will it be practical and feasible for you to sacrifice the cells or tissue required toget this much RNA.?

If not, use as much poly(A) RNA as is practical.


7/62

If using poly(A) RNA in Northern hybridizations.

is the absence of the ribosomal RNA, which are ordinarily usedto gauge the quality and relative quantity of the RNA samplesIn your research.

there are other strategies besides switching to poly(A) RNA that can beused to increase the sensitivity of Northern hybridizations.

You could alter the hybridization conditions of the DNA probe you could switch tousing RNA probes in the hybridization,

RNA probe are( 3 to 5)fold more sensitive than DNA probes in typical hybridizationbuffer.

Dramatic differences in the sensitivity of Northern blots can also be seen fromusing different hybridization buffers.


8/62

Nuclease protection assay.is a technique used to identify individual RNA

molecules in a heterogeneous RNA sample extracted from cells.

The technique can identify one or more RNA molecules of known sequence

even at low total concentration.

RT-PCR (reverse transcription-polymerase chain reaction)

is a sensitive method for the detection of mRNA expression levels.

Traditionally RT-PCR involves two steps:1.the RT reaction .

2.PCR amplification.

RNA is first reverse transcribed into cDNA using a reverse transcriptase .

the resulting cDNA is used as templates for subsequent PCR amplification using

primers specific for one or more genes.


9/62

If you are not interested in determining the size of the target, you can use moresensitive technique such as:1. nuclease protection assay.

2. RT-PCR.

Nuclease protection assays, which are 5- to 10-fold more sensitive than traditionalmembrane hybridizations.

Can accommodate 80 to 100micro gram of nucleic acid in a single

experiment.

RT-PCR can detect extremely rare messages, for example, 400copies of a message in a 1micro gram.

RT-PCR is currently the most sensitive of the RNA analysis techniques.

it is enable to detection and quantitantion of the rarest of targets.


10/62


11/62


12/62


13/62

IF you want quantitative approaches have become increasingly

reliable with introduction of internal standards you must use competitive

PCR strategies.

For quantifying mRNA, internal standard RNAs are added in a defined quantity to the

RNA sample prior to the RT reaction.

The resulting standard cDNA is co amplified with the same primers as the

endogenous target sequence.

Its PCR product is approximately 50 nucleotides smaller.

This method allows measurement of small differences (as low as factor 2) in mRNA

amount between RNA samples.


14/62

Dot slot blotRNA samples are directly applied to a membrane

manually or under vacuum through a filtration manifold.

Hybridization of probe to serial dilutions of sample canquickly generate quantitative data about the expression levelof a target.

Specific sequences in RNA preparations can be detected by blotting

and hybridization analysis using techniques very similar to thoseoriginally developed for DNA.

Fractionated RNA is transferred from anagarose gel to a membrane support(northern blotting).

unfractionated RNA is immobilized by slot or dot blotting.

The resulting blots are studied by hybridization analysis


15/62

Total RNA or poly(A) RNA can be used in this assay since the RNA is not size-fractionated on an agarose gel.

using total RNA in dot/slot blots is that signal of interest cannot bedistinguished from cross-hybridization to rRNA.

Switching to poly(A) RNA as the target source might alleviate this problem.

Hybridization to GeneArraysand Reverse Dot Blots

Gene arrays consist of cDNA clones as a form of oligonucleotides or PCR products.

oligo spotted at high density on a nylon membrane

glass slide, other solid.


16/62

This is difficult to achieve unless poly(A) RNA is used as template in thelabeling reaction.

PCR product by:hybridizing labeled cDNA probes reverse transcribed from mRNA.

the expression of potentially hundreds of genes can be simultaneously

analyzed.

Procedure requires that the labeled cDNA be present in excess of thetarget spotted on the array.


17/62

Ribonuclease Protection Assays

total RNA or poly(A) RNA can be used as starting material

in this assay.

total RNA usually affords enough sensitivity to detect even rare message.

maximum amount ( 80 to 100micro gram).

If the gene is expressed at extremely low levels, requiring weeklongexposure times for detection.

if you want switch to poly(A) RNA might prove beneficial and may justify theadded cost.

This technique is very sensitive,because of do require laborious gelpurification of the full-length probe to avoid getting confusingresults.


18/62

RT-PCRRT-PCR is the most sensitive method for detecting and quantitatingmRNA.

very low-abundance messages can be detected with this technique.

Total RNA is routinely used as the template for RT-PCR.

some cloning situations and rare messages require the use ofpoly(A) RNA

2 different schools of thought concerning RT-PCR.

First ,it advisable to treat the sample RNA with(DNase I )(no purification).

method produces RNA completely free of contaminating genomic DNA.

very small amounts of genomic DNA contamination can cause false positives.A second school of thought avoidance of DNase I.


19/62

cDNA Library Synthesishigh-quality mRNA that is essentially free of ribosomal RNA is required forconstructing cDNA libraries.

Unacceptably high backgrounds of ribosomal RNA clones.

would be produced if total RNA were reverse transcribed to preparecDNA.

cDNA is a DNA copy synthesized from mRNA. T

RNA-dependent DNA polymerase isolated from a retrovirus (AMV or MMLV).

This is provided by the poly(A) tail found at the 3' end of most eukaryotic

mRNAs .

which a short complementary synthetic oligonucleotide (oligo dT primer) is

hybridized .

(polyT -polyA hybrid).


20/62


21/62

Is It Possible to Predict the Total RNA Yield from a Certain Mass ofTissue or Number of Cells?

Sections are based on experimentation at Ambion.

Ambion laboratory technique using a variety of samples and different purificationproducts.

YIEYLD can vary widely based on the type of tissue or cells used for theisolation.

rapid and complete tissue disruption, and homogenizing at subfreezing

temperatures cannot be overemphasized.

because yields from very small amounts of starting material are subject to thelaw of diminishing returns.

choose more starting material rather than less. Samples can be pooled together,if possible, to maximize yields.


22/62

For example, if your sample is5 mg of tissue or 2.5 X 106 cells

You can yields about

10micro g of total RNA,

8microg rRNA,

0.3microg mRNA,1.7micro g tRNA,and other RNA.1 g of tissue or 5X108 cells.

yields about 2mg of total RNA1.6mg rRNA60mg mRNA333mg tRNA and other RNA.


23/62

Protein contamination.Pure RNA has an A260:A280 absorbance ratio of 2.0.

in RNase protection assays.

Northern analysis.

vitro translation experiments.

RT-PCR assays used total RNA with A260:280 ratios ranging from1.4 to 1.8 .

If protein contamination occure is causes to change in these ranges.

Resolving problem is by:

using additional organic extractions with anequal volume of(phenol/chloroform/isoamyl alcohol) (25 : 24: 1mixture) may remove thecontaminant.


24/62

Residual phenol can cause lower the A260:A280 ratio,

inhibit downstream enzymatic reactions.

Chloroform/isoamyl alcohol (24 : 1) extraction will removeresidual phenol.

liver and kidney tissues, produce RNA consistently at a lower absorbancy

ratio than the other absorbancy ratio for these tisuue is rarely above 1.7

Chloroform causes proteins to become denatured and become soluble in the

organic phase or interphase, while nucleic acids remain in the aqueous

phase.

Phenol dissolves proteins and lipids leaving water soluble matter.


25/62


26/62

Physically Intact?

The integrity of your RNA is best determined by electrophoresison a formaldehyde agarose gel under denaturing conditions.

samples can be visualized by adding 10microgm/ml of EtBr to the samplebefore loading on the gel.

The most sensitive test of RNA integrity is Northern analysis using a high molecular

weight probe expressed at low level in the analyzed tissue.

Northern analysis is not tolerant of partially degraded RNA.the quality of the data is severely compromised.

IF a single cleavage in20% of the target molecules will decrease the signal on aNorthern blot by 20%.

Nuclease protection assays and RT-PCR are tolerant.


27/62


28/62

Which Total RNA Isolation Technique Is Most Appropriate forYour Research?

There are three basic methods of isolating total RNA from cellsand tissue samples.

Chaotropic agent use to open the cells and inactivate RNases.

The lysate is then processed in one of several ways to purify the RNA awayfrom protein, genomic DNA, and other cellular components.

These 3 methods include:Guanidium-Cesium Chloride Method

1. Harvest 25 g of tissue, freeze in liquid nitrogen (N2) and grind to

a powder under liquid N2 with a mortar and pestle.

2. Resuspend powder in 150 mL of buffer containing 4.5 M guanidiniumthiocyanate /50 mM EDTA (pH 8.0), 25 mM sodium citrate (pH 7.0), 0.1 M


29/62

2-mercaptoethanol, 2% lauroylsarcosine.

3. Homogenize suspension on amedium setting for 3-5 minutesat 4 C.

4. Remove insoluble material bycentrifuging the suspension in aFiberliteF13-14x50cy carbon fiber rotor at 8,000 xg for 10 minutes at 4 C.

5. Add CsCl to the supernatant at concentration of 0.2 g/ml

6. Layer the sample solution over10 mL of 5.7 M CsCl containing 50 mM EDTA(pH 7.3). If using apolyallomer tube, ensure the tube is completely full and the

5.7 M CsClcushion should occupy one-third of the total tubevolume.ifnecessarytop off tube with 0.2 g/ml CsClbuffered solution.


30/62

7. Centrifuge sample solution inFiberlite F50L-8x39 rotor at

50,000 rpm (~266,000 xg) for 5 hours at 20C.

8. After centrifugation, remove RNA pellet and dissolve in2.0 ml of 10 mM Tris-HCl (pH 7.4), 2 mM EDTA.

9. To further purify, extract RNA3 times with 2 volumes ofphenol/chloroform (1:1) followed by1 extraction with chloroform(Total RNA yield = 3.2 mg).

10. Isolate poly(A)+ RNA by 2 cycles of oligo(dT)-cellulose

chromatography(5) (Final poly(A)+yield = 42 g).


31/62

disadvantageRNA is more dense than DNA and most proteins, it pellets at the bottom of the tubeafter 12 to 24 hours of centrifugation at 32,000rpm.

Small RNAs (e.g., 5S RNA and tRNAs)can not beprepared by this method.

time-consuming, laborious, and required overnight centrifugation.

The number and size of samples that could be processedsimultaneously were limited by the number of spaces in therotor.

Si l d M lti l St G idi A id Ph l M th d


32/62

Single- and Multiple Step Guanidium Acid-Phenol Method

The principle at the basis of the method is that RNA is separated from DNAafter extraction with an acidic solution containing guanidinium thiocyanate,

sodium acetate, phenol and chloroform, followed by centrifugation.

Under acidic conditions, total RNA remains in the upper aqueous phase,while most of DNA and proteins remain either in the interphase or in thelower organic phase.

Total RNA is then recovered by precipitation with isopropanol and can beused for several applications.

The original protocol, enabling the isolation of RNA from cells and tissuesin less than 4 hours, greatly advanced the analysis of gene expressionin plant and animal models as well as in pathological samples.


33/62

why guanidium-acid phenol procedure has largely replaced thecesium cushion method?Because of:

because RNA can be isolated from a large number of samples in two to four hours.

the technique can be easily scaled up or down to process differentsample sizes.

is based on the propensity of RNA molecules to remain dissolved in the

aqueous phase in a solution containing 4M guanidium thiocyanate.ph4

DNA remain in the organic phase.

proteins and other cellular macromolecules are retained at the interphase.


34/62

Problem during the phenol/chloroform extraction step

The mixture must be spun with sufficient force to ensure adequate separation of theorganic and aqueous layers. depend on the rotor.

The interface between the aqueous and organic layers is another potential sourceof genomic contamination.

Avoid the white interface (cream colored or brownish) between the two layers;

leave some of the aqueous layer with the organic layer.

Residual salt from the precipitation step, appearing as a hugewhite pellet, can interfere with subsequent reactions.

Excess salt can be removed by washing the RNA pellet with 70% EtOH (ACS

grade).


35/62

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes

ethanol removed by aspiration with a drawn out Pasteur pipet after re spun.


36/62


37/62

Non-Phenol-Based Methods (phenol-free methods).

Properities:Very fast, clean RNA, can process large sample numbers.

Free from the handling and disposal of phenol, a very hazardouschemical.yields total RNA of the same quality as the phenol-based methods.

based on the ability of glass fiber filters to bind nucleic acids in the presence ofchaotropic salts like guanidium,

requiring no organic extractions.

processing large sample numbers is fast and easy. the quickest methodsfor RNA isolation completed in less than one hour


38/62

Problems.DNA contamination can be higher with this method than withphenol-based methods.

The primary problem is clogging of the glass fiber filter by thick lysates. This canbe prevented by using a larger volume of lysis buffer initially.

frothing amass of bubbles in or on a liquid foam.

Second problem is to minimize the viscosity of the lysate bysonication.(on ice, avoid power settings that generate frothing) .

If your sample is enriched by high in saccharides or fatty acidsan initial clarifying spin or extraction with an equalvolume of chloroform can prevent filter-clogging problems.


39/62

You can solve by clarifying spin is 8 minutes at(7650 Xg.).

if you failed in this solving the lysate can be divided into micro centrifuge tubesand centrifuged at maximum speed for 5 to 10 minutes.

if your sample is rich in glycogen such as liver, or plants containing highmolecular weight carbohydrates.

Avoid initial clarifying spins on tissues.

If you generate a clogged filter, remove the remainder of the lysate using apipettor.

place it on top of a fresh filter, and continue with the isolation protocol usingboth filters.


40/62

steps:1.the cells are first lysed in a guanidium-based buffer.

2.The lysate is then diluted with an organic solvent such as ethanol or

Isopropanol.

3.applied to a glass fiber filter or resin.

4.DNA and proteins are washed off, and the RNA is eluted at the end in anaqueous buffer.


41/62

What Protocol Modifications Should Be Used for RNA Isolationfrom Difficult Tissues?

Protocol modification necessary to eliminate specific contaminants, or tissuetreatments prior to the RNA isolation protocol.like

Fibrous tissues, tissue rich in protein, DNA and RNases,

Type of sample cells and tissues.Fibrous Tissue.Like heart and muscle

Due to low cell density and the poly nucleate nature of muscle tissue

You do it pulverizing the frozen tissue into a powder while keeping the tissuecompletely frozen (use a chilled mortar and pestle) is the key to isolating intacttotal RNA.


42/62

Lipid and PolysaccharideRich TissueSuch as Plant and brain tissues.

difficult to get clean separation of the RNA

If you use phenol-based methods to isolate total RNA,white flocculent material present through out the aqueous phase.

This flocculate will not accumulate at the interface.

Chloroform: isoamyl alcohol (24 : 1) extraction of the lysate is probably thebest way to partition the lipids away from the RNA.

To minimize loss, back-extract the organic phase, and clean up therecovered aqueous RNA by extraction with phenol : chloroform : isoamylalcohol (25 : 24:1).


43/62

When your sample is plant tissue using a non-phenolbasedmethod,polyvinylpyrrolidone-40 (PVP-40) can be added to the lysate to absorbpolysaccharide and polyphenolic contaminants

.When the lysate is centrifuged to remove cell debris, thesecontaminants will be pelleted with the PVP

Centrifugation on cesium tri fluoroacetate has also been shown to separatecarbohydrate complexes from RNA

The use of cesium trifluoroacetate, rather than cesium chloride, for density

gradient centrifugation improves both the yield and purity of total RNA .


44/62


45/62

Nucleic Acid and Nuclease-Rich Tissue

Spleen and thymus

Best homogenization is the key to isolating high-quality RNA from thesetissues.

samples should be completely pulverized on dry ice, under liquid nitrogen, tofacilitate rapid homogenization in the lysis solution, and cause inhibitsnucleases.

If your sample is Cancerous cells contain high amounts of DNA and RNA.

,These cells are unusually viscous.causing poor separation of the organic andaqueous phases and potentially clogging RNA binding filters.

Increasing the ratio of sample mass : volume of lysis buffer can helpalleviate this problem in filter-based isolations.


46/62

How can you resolve this problem?

Increasing the ratio of sample mass : volume of lysis buffer can help alleviate thisproblem in filter-based isolations.

Multiple acidphenol extractions can be done to ensure that most of the DNA ispartitioned into the organic phase during acid phenol based isolation procedures.

If a lysate of your sample is viscous by attempting to pipet the solution and

observing whether it sticks to the pipette tip.

The DNA in the lysate can alternatively sheared.

by vigorous and repeated aspiration through a small gauge needle (18 gauge) orby sonication (10 second sonication at 1/3 maximum power on ice, or until the

viscosity is reduced.


47/62


48/62

Hard Tissue


49/62

Hard Tissue

bone and tree bark.

disrupted using a PolytronTM or any other commonly available homogenizer.

Heavy-duty tissue grinders that pulverize the material using mechanicalforce are needed. SPEX Certiprep.

Tissue-grinding mills that chill samples to liquid nitrogen temperatures andpulverize them by shuttling a steel piston back and forth inside a stationarygrinding vial.


50/62

Bacterial and yeast cellsquite refractory to isolating good-quality RNA due to the difficulty of lysingthem

bacteria is the short half-life of most bacterial messages.

Lysis can be facilitated by re suspending cell pellets in TE and treatingwith lysozyme

TE use to solubilize the RNA and protect from the degredation.

They can introduce RNases. use the highest quality enzymes to reduce theof introducing contaminants.

You can obtainYield and quality by use phenol-based extraction protocols can

be improved by conducting the organic extractions at high temperatures.


51/62


52/62

Yeast cells is accomplished by vigorous vortexing in the presence of 0.4 to 0.5 mmglass beads.

Yeast cells can be treated by zymolase, lyticase, and glucolaseto lysis.

Poly(A) RNA Purification Strategies.

purify poly(A) RNA directly from the starting material via by onestep strategy.

first isolates total RNA, then purifies poly(A) RNA from that by 2steps strategy.

These 2 different steps are difference in number, size of sample andyield produce.

Making and using mRNA (2)


53/62


54/62

One-step strategies involve fewer manipulations to recover poly(A) RNA

Two additional washing steps multiplied by 20 samples

consume significant time and materials, and arguably, faster.

purification strategies decrease the chance of degradation

The true speed of a technique is determined by the total manipulations in a

procedure.

one step procedures is used in hybridization of gene array.

Percentage of poly(A) RNA is similar between one- and two-step strategies.

But when experimental sample is limited, a one-step procedure is the morepractical procedure.


55/62

one-step products are usually geared to purify small (15microg) or large(25microg) quantities of poly(A) RNA.

If you require more poly(A) RNA, a two-step procedure is usually more cost

effective.

RNase-Free Technique

RNase contamination is prevalent,

You must be prepare a solutions to dissolve problem should be prepared indisposable.

RNase-free plastic ware .

RNase-free glassware prepared in the lab.

Glassware: by baking RNaseat 180C for 8 hours to overnight.

Treating with a commercial RNase decontaminating solution.

Also you can solve Rnase problem by:


56/62

Also you can solve Rnase problem by:

RNase can be removed by filling containers with 0.1% DEPC.

incubating at 37C for 2 hours.

Rinsing with sterile water .

Heating to100C for 15 minutes.

Autoclaving for 15 minutes to eliminate RNase.

Electrophoresis apparatus used for RNA analysis can be made:

RNase-free by filling with a 3% hydrogen peroxide solution.

Incubation period 10 minutes at room temperature.

Rinsing with DEPC-treated water.


57/62

DEPC inactivates RNases by carboxyethylation of specific amino acid sidechains.

DEPC is a suspected carcinogen.be careful with handling it.

this inactivates the residual DEPC by hydrolysis causes to

Releases CO2

EtOH as by-products

The EtOH by-product can combine with trace carboxylic acid

contaminates in the vessel to form volatile esters.

increasing the concentration of DEPC to 1% can inhibit more RNase but can alsoinhibit certain enzymatic reactions,is usually not better.

Diethyl pyro carbonate.?how it act ?

DEPC-Treated Water


58/62

It should be noted that many reagents commonly used in RNA studies containprimary amines, such as Tris, MOPS,HEPES, and PBS, and cannot be DEPC-treated because the amino group sops up the DEPC.

making it unavailable to inactivate Rnase.

MOPS(N-morpholino)propane sulfonic acid,

HEPES(4-2-hydroxyethyl)-1-piperazineethanesulfonic acid)

PBS: phosphate buffered saline


59/62

How Do You Minimize RNA Degradation during SampleCollection and Storage?

In other experiments homogenized tissue has been stored for at least oneweek at room temperature or two months at 4C without any loss of RNA in alysis buffer .

A commercial RNase inhibitor also exists that can prevent RNA degradationwithin mammalian tissue, cells, and plant tissues stored above freezingtemperature for long periods.

RNase is present in all cells and tissues they must be immediately inactivatedwhen the source organism dies. Samples should be immediately frozen inliquid nitrogen.

Safe Place to Pause during an RNA Purification Procedure


60/62

Ideally RNA should be purified without interruption.

stop when the RNA is precipitated or is in the presence of a chaotrope.

For example, when using an organic isolation procedure, the RNA isolation canbe stopped when the samples have been homogenized in a chaotrophiclysis solution.

They can be stored for a few days at -20C or -80C without degradation.

Safe Place to Pause during an RNA Purification Procedure.


61/62

short-term storage

a few weeks or less, store your RNA in RNase-freeTris-EDTA or 1mM EDTAat -20C in aliquots.

Long term storage.RNA should be stored in aliquots at -80C in TE,1mM EDTA, formamide, or asan ethanol/salt precipitation mixture.

STORAGE OF PURIFIED RNA.


62/62

Presentation 33333

Documents