real-time polymerase chain reaction

Real-time polymerase chain reaction

Presented by: Farhad Jahanfar

Introduction to PCR

PCR was invented in 1984 by ( Kary mullis ) & he

received the Nobel Prize in chemistry in 1993, for

his invention.(1)

It revolutionized biological methods specially in

molecular cloning in a way that it has

became an inseparable & irreplaceable part of

molecular investigations.

2

What you need for PCR• Together in a reaction tube on;

– Sample (+/- target DNA)

– Primers for the specific detection

– Nucleotides (dNTPs)

– Enzyme (taq polymerase, pfu or...)

– Buffer

– MgCl2

– Water

– Addetives( optional)Ficoll400 and tartrazine omit the need for gel loading buffer in both agarose and PAGE.

– Extras(enhancers)

Extraction Methods1) Organic (phenol/chloroform)

2) Non-Organic (Proteinase K and Salting out)

3) Chelex: styrene di-vinylbenzene copolymer containing paired imino –di-acetate ions

4) FTA Paper

5) Boiling

• Note: The method utilized may be sample dependant, techniquedependant, or analyst preference

DNA template troubleshooting

• DNA Template• Usually,100ng is

sufficient for a good PCR product (1 ngr-1 µgr).

• Amount of DNA present▫ Less DNA means more cycles, high means false

priming and • Complexity of DNA

▫ Ex. plasmid vs. whole genome• Purity

▫ Interfering factors, eg. enzymes, salts▫ If salting out was used in Extraction may

contamination by salts.• Degradation

▫ PCR more forgiving of degraded DNA• Contamination: urea, SDS, Na acetate

▫ Addition organic extractions, ethanol precipitation orPAGE Vs. agarose purification will decrease suchcontamination.

• Presence of “poisons”▫ Eg. EDTA which scavenges Mg 2+

Primer troubleshooting

• Age

• Number of freeze-thaws

• Contamination

• Amount

– Can vary over a wide range (50X)

– 100-500 nM typical( 0.1 -1 μm each primer)

– Too low: low amplification

– Too high: low amplification or unspecific bands, primer dimers

– Comment; use primers without complementary specially in 3’end.

– For short DNA fragments (100bp) may need more primers, ex;>1

μm of each primers

Buffer Considerations

• Most reaction buffers consist of a buffering agent, most

often a Tris-based buffer, and salt, commonly KCl.

• The buffer: regulates the pH of the reaction (affects the

DNA polymerase activity and fidelity).

• Modest concentrations of KCl will increase DNA

polymerase activity, by 50–60% over activities in the

absence of KCl; 50mM KCl is considered optimal.

•

Buffer Considerations• DNA Polymerase contains native Taq DNA polymerase in a

proprietary formulation. It is supplied with 5X GreenGoTaq® Reaction Buffer and 5X Colorless GoTaq.

5X Green GoTaq® Reaction Buffer: contains 2 dyes (blueand yellow) that separate during electrophoresis tomonitor migration progress and also contains a compoundthat increases the density of the sample to sink into thewell of the agarose gel, allowing reactions to be directlyloaded onto an agarose gel, No the need for loading dye.

The blue dye comigrates at the same rate as a 3–5kb DNAfragment in a 1% agarose gel.

The yellow dye migrates at a rate faster in a 1% agarosegel.

Buffer troubleshooting• Buffer

• To avoid contamination must aliquot insmall cap-tubes (avoid nucleasecontamination).

• Using high quality reagents.• Must match to polymerase.• Typically contain KCl and Tris.• Can vary over a slight range:

▫ Not much difference in range from 0.8 X to 2.0 X

▫ With or without dye.▫ Note: Primer efficiency reduced outside

this range.

Mg2+ concentration•Magnesium is a required cofactor for Taq DNA

polymerases, and Mg2+concentration is a crucial

factor that can affect amplification success.

• Template DNA concentration, chelating agents

present in the sample (e.g., EDTA or citrate),

dNTPs concentration and the presence of

proteins can all affect the amount of free Mg2+ in

the reaction.

•

•Figure: different amounts of free Mg2+, TaqDNA polymerase is inactive

Mg2+ concentration Excess free Mg2+, reduces enzyme fidelity and may

increase the level of nonspecific amplification.

So, empirically determine the optimal magnesiumconcentration for each target.

The effect of Mg2+ concentration and the optimalconcentration range can vary with the particular DNApolymerase. For example, the performance of Pfu DNApolymerase seems to be less dependent on Mg2+concentration.

Optimization is required, the optimal concentration isusually in the range of 2–6mM.

Mg2+ concentration• Many DNA polymerases are supplied with a Mg-free

reaction buffer and a tube of 25mM MgCl2 so that youcan adjust the Mg2+ concentration to the level that isoptimal for each reaction.

• Comment: Before assembling the reactions, be sure tothaw the magnesium solution completely prior to useand vortex the magnesium solution for several seconds(to obtain a uniform solution) before pipetting.

• .

• MgCl2 • Mg2+ is an essential cofactor of DNA polymerase and need for dsDNA establishment.

• Amount can vary:

– 0.5 to 3.5 μm (1-4μm) suggested

– Too low: Taq won’t work

– Too high: mispriming, PCR fidelitydecreasing, un-specific PCRproducts ( ladder formation orsmear)

dNTPs troubleshooting

• Nucleotides20-400 μM (10-200 each)works well

▫ - 200mM of each is enough to synthesize 12.5mg of DNA.

▫ Too much: can lead to mispriming and errors if >200mM each increase the error rate of polymerase.

▫ Too much: can scavenge Mg2+

▫ Too low: faint productsAgeNumber of freeze-thawsDilute in buffer (eg. 10mM Tris pH 7.4 -7.5 to

prevent acid hydrolysis)ContaminationFor long fragments need higher

dNTPsHigh change in dNTPs amount need

adjust of Mg2+

Enzyme Concentration

using 1–2.5 units of Taq DNA polymerase in a 50μl

amplification reaction, is recommended.

In most cases, an excess of enzyme, will not significantly

increase product yield.

In fact, increased amounts of enzyme increase the

likelihood of generating artifacts associated with the

intrinsic 5′→3′ exonuclease activity of Taq DNA

polymerase, resulting in smeared bands in an agarose gel.

Note: Pipetting errors are a frequent cause of excessive

enzyme levels.

• Taq Polymerase• Error rate 2x10-4N/C

• Thermostable! Activity declines with time at 95₀C(Hot start)

• Matches buffer• Age• Contamination• Concentration: Typically 0.5 to 1.0 U/for

a 25 μl reaction.

• If low; incompleteelongation(defective PCR products)

• If high: un specific bands and smearformation in gel.

• Note: usually adding 2.5 U/reactionMAY INCREASE but up to the point.

A Typical PCR Reaction

Sterile Water 38.0 ul

10X PCR Buffer 5.0 ul

MgCl2 (50mM) 2.5 ul

dNTP’s (10mM each) 1.0 ul

PrimerFWD (25 pmol/ul) 1.0 ul

PrimerREV 1.0 ul

DNA Polymerase 0.5 ul

DNA Template 1.0 ul

Total Volume 50.0 ul

Cycling• Cycling parameters: each step requires a minimal

amount of time to be effective, while too much timecan be both wasteful and deleterious to polymerase.

• Annealing step: Primers with low GC% needtemperature lower than 55°C.

• Extention step: temperature is usually 72°C, 1 min/kbproduct length is sufficient.

• Ramp time: the time it takes to change from onetemperature to another.

Choosing cycling parameters• Usual program

• 30 cycles: 30 sec 94ºC denaturation

• ---------------------------------------------------------------------------------------------

• 30 sec 55ºC (GC content≤50%) or annealing

• 60 ºC (GC content>50%)

• ------------------------------------------------------------------------------------------------

• 72ºC extension

.

Cycling is dependent upon

The sequence and length of the template DNA,

The sequence and complexity of primers,

The ramp times of the thermal cycler used,

The number of cycles depend on both the efficacy of reaction

and the amount of template DNA.

Note: Greater cycles No. (>40) can reduce the Taq efficacy and

increase the nonspecific bands and deplete substrate.

PCR Cycling Parameters

• Denaturation Step Must balance DNA denaturation with Taq damage.

95₀C for 30 - 60s typically is enough to denature DNA.

Even 92 ₀ C for 1s can be enough.

Taq loses activity at high temps:▫ Half-life at 95 ₀ C: 40 min

▫ Half-life at 97.5 ₀ C: 5 min


• Annealing Step

• Most critical step

• Calculate based on Tm.

Comment: Using an annealing temperature slightlyhigher than the primer Tm will increase annealingstringency and can minimize nonspecific primerannealing decrease the amount of undesired productssynthesized

▫ Often does not give expected results• Trial-and-Error

▫ Almost always must be done anyway

▫ Too hot: no products

▫ Too low: non-specific products

Comment: Gradient thermocyclers are very useful

• Typically only 20s needed for primers to anneal

PCR Cycling Parameters• Extension Step

• Temperature typically 72 ₀ C

– Reaction will also work well at 65₀ C or other temps

• Time (in minutes) roughly equal tosize of the largest product in kb

– Polymerase runs at 60bp/sunder optimum conditions

• Final “long” extension step mostlyunnecessary


• Number of Cycles • Source of DNA molecules:– >100,000: 25-30 cycles

– >10,000: 30-35 “

– >1,000: 35-40 “

– <50: 20-30 fb. nested PCR

• Do not run more than 40 cycles.– Virtually no gain

– Extremely high chance of non-specific products

• Best optimized by trial-and-error

Comment: If nonspecific amplification products accumulate, diluting the

products of the first reaction and performing a 2th amplification with the same

primers or primers that anneal to sequences within the desired PCR product

(nested primers).

Basic Experimental Design

• A well-designedexperiment can keep youfrom ever getting intotrouble!

Basic Experimental Design Comments

• Main point: Always use CONTROLS

• Positive control– So you’ll know what a

successful result looks like.

• Negative control– Lets you know if you have

contamination.

Experimental Design: Controls

No positive or negative controls…What does this result mean??

Only a positive control…How do we know the result isn’t due tocontamination?

Both positive and negative controls…Results can be interpreted withconfidence.

U

U +

U - +

Basic Experimental Design Comments

If no band , 1th add 10 cycles

Dilute 1th PCR product and re PCR

Increase the denaturation temperature

Add enhancers

Using TAE in Electrophoresis Vs. TBS can delete

the smears.

Basic Experimental Design PCR Cycling Parameters

• “Odd” Protocols • Hot-Start PCR

– Taq is added latter

• Touchdown PCR

– Annealing temperature is progressively reduced.

Extras troubleshooting

•Higher yields can achieve by:• A) By using Noninonic detergents (Triton X-100) neutrialize

charges of ionic detergents often use in DNA extraction.

• B) By using Taq DNA Polymerase stabilizing activity with

enzyme stabilizing proteins (BSA or gelatin)

• C) By using Polymerase stabilizing solutes (betain)

• D) By using Enzyme stabilizing solvents (glycerol)

• E) By using Solubility Enhancing solvents (DMSO)

• F) By using Molecular crowding solvents (PEG)

• G) By using Polymerase salt preference( NH4SO4)

Cont’d Greater specificity is achieved by:

Lowering Tm of dsDNA (by Formamide); lowers meltingtemperatures (Tm) of DNAs linearly by 2.4-2.9 C/mole offormamide, depending on the (G+C) composition, helixconformation and state of hydration.

Destabilizing mismatched primer annealing (by PMPE* or Hotstart strategies)

Amplification of high GC components by:

by using betain*

Other PCR Reaction Components

• Extras=

• enhancers

Added by user:

Glycerol(5-20%), DMSO(1-10%)Stabilize Taq, decrease secondary structure

Probable help or hurt, depending on primers

Typically already in the Taq stock

Betaine (1-2 M)Useful for GC-rich templates

For long templates, higher pH isrecommended(9.0), the pH of Tris bufferdecreases at high temperatures, long-templatePCR requires more time at high temperatures andincreasing time at lower pH may cause somedepurination of the template resulting in reducedyield of specific product.

Cont’d

PEG 6000(5-15%)

Non ioning detergents

Foramide (1.25%-10%), DMSO (1–10%) or BSA (10-100 μg/ml)

which frequently helps, doesn’t hurt.

Note: Concentrations of DMSO greater than 10% and

formamide greater than 5% can inhibit Taq DNA

polymerase and presumably other DNA polymerases as

well.

Analyze the productelectrophoresis

• Agarose

Analyze the productelectrophoresis

• PAGE

Analyze the product Electrophoresis 10µl from each reaction on an agarose

and nondenaturing PAGE, for resolution of PCR products

between 100 to 1000bp.

Further staining by Ethidium bromide (Etbr) or SYBR (25

to 200) times more sensitive than Etbr, more convenient

to use, and permits optimization of 10 to 100 fold lower

starting template copy No. Silver staining in PAGE (

more sensitive)

What do mRNA levels tell us?

DNAmRNAprotein

• Reflect level of gene expression

• Information about cell response

• Protein production (not always)

quantitative mRNA/DNA analysis

Direct

-Northern blotting

-In situ hybridization

PCR amplification

-Regular RT-PCR

-Real time PCR

(Microarrays)

Nomenclature

RT-PCR = Reverse Transcriptase PCR

qReal time PCR = quantitative Real-Time PCR

RT-PCR

• Isolate RNA

• cDNA synthesis

• PCR reaction

Annealing of Downstream Primer to RNA

Reverse Transcription With AMV Reverse Transcriptase

RNA Copied From 3’ to 5’ into cDNA

Amplification of cDNA by PCR

Why isn´t this good enough?

What’s Wrong With

Agarose Gels?

* Low sensitivity

* Low resolution

* Non-automated

* Size-based discrimination only

* Results are not expressed as numbers

based on personal evaluation

• Ethidium bromide staining is not very quantitative

• End point analysis

Different concentrations give similar endpoint results!

Endpoint analysis

How does real-time PCR work?

To best understand what real-time PCR

is, let’s review how regular PCR

works...

The Polymerase Chain Reaction

How does PCR work??5’

5’

3’

3’

d.NTPs

Thermal Stable DNA Polymerase

Primers

Denaturation

Annealing

Add to Reaction Tube


How does PCR work??

Extension

5’ 3’

5’3’

Extension Continued

5’ 3’

5’3’

Taq

Taq

3’

5’3’

Taq

Taq

Repeat


How does PCR work??

5’3’

3’

3’

3’

5’3’3’

5’3’

3’Cycle 2

4 Copies

Cycle 3

8 Copies

3’

3’

5’3’

3’

5’3’

3’

5’3’

3’

5’3’

3’

5’3’3’

5’3’

3’

5’3’

3’

Imagining Real-Time

PCR

…So that’s how PCR is usually presented.

To understand real-time PCR, let’s imagine

ourselves in a PCR reaction tube at cycle

number 25…

Imagining Real-Time

PCR

What’s in our tube, at cycle number 25?

A soup of nucleotides, primers, template,

amplicons, enzyme, etc.

1,000,000 copies of the amplicon right now.

Imagining Real-Time

PCR

How did we get here?

What was it like last cycle, 24?

Almost exactly the same, except there were only 500,000 copies of the amplicon.

And the cycle before that, 23?

Almost the same, but only 250,000 copies of the amplicon.

And what about cycle 22?

Not a whole lot different. 125,000 copies of the amplicon.

Imagining Real-Time PCR


If we were to graph the amount of DNA in our

tube, from the start until right now, at cycle

25, the graph would look like this:

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

0 5 10 15 20 25 30 35 40



So, right now we’re at cycle 25 in a soup with

1,000,000 copies of the target.

What’s it going to be like after the next cycle,

in cycle 26?

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

0 5 10 15 20 25 30 35 40

?


So where are we going?

What’s it going to be like after the next cycle, in cycle 26?

Probably there will be 2,000,000 amplicons.

And cycle 27?

Maybe 4,000,000 amplicons.

And at cycle 200?

In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…

Or 10^35 tonnes of DNA…

To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!

Imagining Real-Time

PCR


A clump of DNA the size of ten billion planets

won’t quite fit in our PCR tube anymore.

Realistically, at the chain reaction progresses,

it gets exponentially harder to find primers,

and nucleotides. And the polymerase is

wearing out.

So exponential growth does not go on

forever!



If we plot the amount of DNA in our tube

going forward from cycle 25, we see that it

actually looks like this:

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

MeasuringQuantities

How can all this be used to measure DNA

quantities??

What if YOU started with FOUR times as much

DNA template as I did?

I have 1,000,000 copies at cycle 25.

You have 4,000,000 copies!

So… You had 2,000,000 copies at cycle 24.

And… You had 1,000,000 copies at cycle 23.

Imagining Real-Time

PCR

MeasuringQuantities

So… if YOU started with FOUR times as much

DNA template as I did…

Then you’d reach 1,000,000 copies exactly

TWO cycles earlier than I would!

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

MeasuringQuantities

What if YOU started with EIGHT times LESS DNA template

than I did?

You’d only have 125,000 copies right now at cycle 25…

…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle

28 you’ll have caught up with 1,000,000 copies!

So… you’d reach 1,000,000 copies exactly THREE cycles

later than I would!

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

MeasuringQuantities

We describe the position of the lines with a value that

represents the cycle number where the trace crosses

an arbitrary threshold.

This is called the “Ct Value”.

Ct values are directly related to the starting quantity of

DNA, by way of the formula:

Quantity = 2^Ct

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

23 25 28

Ct Values:

67

Imagining Real-Time

PCR

MeasuringQuantities

There’s a DIRECT relationship between the

starting amount of DNA, and the cycle

number that you’ll reach an arbitrary

number of DNA copies (Ct value).

DNA amount = 2 ^ Cycle Number

C o p y N u m b e r v s. C t - Sta n d a r d C u r v e

y = -3 . 3 1 9 2 x + 3 9 . 7 7 2

R2 = 0 .9 9 6 7

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1

Lo g o f co p y n u m b er (1 0 n )

Ct

Real-time PCR advantages

* not influenced by non-specific amplification

* amplification can be monitored real-time

* no post-PCR processing of products(high throughput, low contamination risk)

* requirement of 1000-fold less RNA than conventional assays(3 picogram = one genome equivalent)

* most specific, sensitive and reproducible

Real-time PCR disadvantages

* setting up requires high technical skill and support

* high equipment cost

* Runs are more expensive than conventional PCR

* DNA contamination (in mRNA analysis)

Cycle Threshold

* cycle threshold or the CT value is the cycle at which a significant increase in DRn is first detected

* it is the parameter used for quantitation

* CT value of 40 or more means no amplification and cannot be included in the calculations

Data analysis

PCR phases in linear view

Cycle #

[DNA]

PCR phases

Exponential◦ If 100% efficiency – exact doubling of products.

Specific and precise

Linear◦ High variability. Reaction components are being

consumed and PCR products are starting to degrade.

Plateau◦ End-point analysis. The reaction has stopped and if left

for long – degradation of PCR products.

72

more…

Exponential

Linear

Plateau

PCR buffer

dNTP Mix

Thermostable DNA polymerase

Template

DDW

…

73

74

Gene of interest

Mutation Detection

Allelic discrimination

Gene expression ( mRNA)

Microbial agents detection

Quantification

…Disease

General rules for primer design

-- Specificity and cross homology

Specificity

Determined primarily by primer length as well as sequence

The adequacy of primer specificity is dependent on the nature of the

template used in the PCR reaction.

Cross homology

Cross homology may become a problem when PCR template is genomic

DNA or consists of mixed gene fragments.

Primers containing highly repetitive sequence are prone to generate non-

specific amplicons when amplifying genomic DNA.

Avoid non-specific amplification

BLASTing PCR primers against NCBI non-redundant sequence database

is a common way to avoid designing primers that may amplify non-

targeted homologous regions.

Primers spanning intron-exon boundaries to avoid non-specific

amplification of gDNA due to cDNA contamination.

Primers spanning exon-exon boundaries to avoid non-specific amplification

cDNA due to gDNA contamination.


-- Primer and amplicon length

Primer length determines the specificity and

significantly affect its annealing to the template

Too short -- low specificity, resulting in non-specific

amplification

Too long -- decrease the template-binding efficiency at

normal annealing temperature due to the higher probability

of forming secondary structures such as hairpins.

Optimal primer length

18-24 bp for general applications

30-35 bp for multiplex PCR

Optimal amplicon size

300-1000 bp for general application, avoid > 3 kb

50-150 bp for real-time PCR, avoid > 400 bp


-- Melting temperature (Tm)

Tm is the temperature at which 50% of the DNA duplex

dissociates to become single stranded

Determined by primer length, base composition and concentration.

Also affected by the salt concentration of the PCR reaction mix

Working approximation: Tm=2(A+T)+4(G+C) (suitable only for 18mer

or shorter).

Optimal melting temperature

52°C-- 60°C

Tm above 65°C should be generally avoided because of the potential for

secondary annealing.

Higher Tm (75°C-- 80°C) is recommended for amplifying high GC

content targets.

Primer pair Tm mismatch

Significant primer pair Tm mismatch can lead to poor amplification

Desirable Tm difference < 5°C between the primer pair


-- Annealing temperatures and other considerations

Ta (Annealing temperature) vs. Tm

Ta is determined by the Tm of both primers and amplicons:

optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25

General rule: Ta is 5°C lower than Tm

Higher Ta enhances specific amplification but may lower yields

Crucial in detecting polymorphisms

Primer location on template Dictated by the purpose of the experiment

For detection purpose, section towards 3’ end may be preferred.

When using composite primers Initial calculations and considerations should emphasize on the template-

specific part of the primers

Consider nested PCR


-- GC content; repeats and runs

Primer G/C content

Optimal G/C content: 45-55%

Common G/C content range: 40-60%

Runs (single base stretches)

Long runs increases mis-priming (non-specific annealing)

potential

The maximum acceptable number of runs is 4 bp

Repeats (consecutive di-nucleotide)

Repeats increases mis-priming potential

The maximum acceptable number of repeats is 4 di-

nucleotide


-- Primer secondary structures

Hairpins

Formed via intra-molecular interactions

Negatively affect primer-template binding, leading to poor or no

amplification

Acceptable ΔG (free energy required to break the structure): >-2

kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;

Self-Dimer (homodimer)

Formed by inter-molecular interactions between the two same primers

Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for

internal self-dimer;

Cross-Dimer (heterodimer)

Formed by inter-molecular interactions between the sense and antisense

primers

Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for

internal cross-dimer;


-- GC clamp and max 3’ end stability

GC clamp

Refers to the presence of G or C within the last 4 bases from

the 3’ end of primers

Essential for preventing mis-priming and enhancing specific

primer-template binding

Avoid >3 G’s or C’s near the 3’ end

Max 3’end stability

Refers to the maximum ΔG of the 5 bases from the 3’end of

primers.

While higher 3’end stability improves priming efficiency, too

higher stability could negatively affect specificity because of

3’-terminal partial hybridization induced non-specific

extension.

Avoid ΔG < -9.

Primer Design Considerations

Consideration Comment

Primer Length 18-30 bases

Primer Melting Temperature (Tm) 55°-72°C

Primer Annealing Temperature (Ta) ~5°C < the lowest Tm of the of primers

Tm difference between forward and reverse primers ≤ 5°C

Max 3′ Stability ∆G value for five bases from 3′ end

Percentage GC content 40-60%

No Secondary StructuresIdentify primer pairs which do not assume

secondary structure

No self-complementarity < 4 contiguous bases

No complementarity to other primer(s) < 4 contiguous bases

No long runs with the same base < 4 contiguous bases

Distance between two primers on target sequence < 2000 bases apart

Plateau Effect accumulation of product ≤0.3 to 1 pmol

83

Tool name URL

CODEHOP http://blocks.fhcrc.org/codehop.html

Gene Fisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/

DoPrimer http://doprimer.interactiva.de/

Primer3 http://frodo.wi.mit.edu/primer3/

Primer Selection Http://alces.med.umn.edu/rawprimer.html

Web Primer http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer

PCR designer http://cedar.genetics.ston.ac.uk/public_html/primer.html

Primo pro 3.4 http://www.changbioscience.com/primo.html

Primo Degenerate

3.4

http://www.changbioscience.com/primo/primod.html

PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer

The Primer

Generator

http://www.med.jhu.edu/medcenter/primer/primer.cgi

EPRIMERS http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html

PRIMO http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3

PrimerQuest http://www.idtdna.com/biotools/primer_quest/primer_quest.asp

MethPrimer http://itsa.uscf/~uralab/methprimer/index1.html

Rawprimer http://alces.med.umn.edu/rawprimer.html

MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/

The Primer Prim’er

Project

http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj

ect/primer.html

GAP http://promoter.ics.uci.edu/primers/

84

Description Software name

Analyses a template DNA sequence and chooses primer pairs for PCR and

primers for DNA sequencing

Primerselect

DANASIS Max is a fully integrated program that includes a wide range of

standard sequence analysis features.

DANSIS Max

Primer design for windows and power macintosh. Primer Primer 5

Comprehensive primer design for windows and Power Macintosh. Primer Primer:

Comprehensive analysis of individual primers and primer pairs. NetPrimer

For fast, effective design of specific oligos or PCR primer pairs for microarrays. Array Designer 2

Design molecular beacons and TaqMan probes for robust amplification and

fluorescence in real time PCR.

AlleleID 7

Primer design for DNA-arrays/chips. GenomePRIDE 1.0

Software for Microsoft Windows has specific. Ready-to-use template for many

PCR and sequencing applications; standard and long PCR inverse PCR.

Degenerate PCR directly on amino acid sequence. Multiplex PCR.

Fast PCR

Primer Analysis Software for Mac and Windows. OLIGO 7

Will find optimal primers in target regions of DNA or protein molecules, amplify

leatures in molecules, or create products of a specified length.

Primer Designer 4

Software for primer design. GPRIME

Genome Oligo Designer is a Software for automatic large scale design of optimal

oligonucleotide probes for microarray experiments.

Sarani Gold

Primer and template design and analysis. PCR Help

Genorama Chip Design Software is a complete set of programs required for

genotyping chip design.The programs can also be bought separately.

Genorama chip Design

Software

The Primer Designer features a powerful, yet extremely simple, real-time interface

to allow the rapid identification of theoretical ideal primers for your PCR

reactions.

Primer Designer

Automatic design tools for PCR. Sequencing or hybridization probes, degenerate

primer design, restriction, Nested/Multiplex primer design, restriction enzyme

analysis and more.

Primer Primer

DOS-program to choose primer for PCR or oligonucleotide probes. PreimerDesign

85

86

87

88

Mature RNA

Primary transcript

89

Exon 1 Exon 3Exon 2

This primer spans the exon 2- exon3 junction

Exon 1 Exon 2 Exon 3

90

91

Imagining Real-Time

PCR

What’s in our tube, at cycle number 25?

A soup of nucleotides, primers, template,

amplicons, enzyme, etc.

1,000,000 copies of the amplicon right now.

Intercalating Dyes ( SYBR GreenІ )

Hydrolysis probes ( TaqMan )

Molecular Beacons

FRET probes

Scorpions

AND …

General methods for Real time PCR detection

93

5’

5’

3’

3’

d.NTPs


Primers Add Master Mix and Sample

Denaturation

Annealing

Reaction Tube

SYBR GreenІ

Intercalation Dyes

Taq ID

l

Extension

5’ 3’

5’3’

Extension ContinuedApply ExcitationWavelength

5’ 3’

5’3’

Taq

Taq

3’

5’3’

Taq

Taq

Repeat

SYBR GreenІ

ID ID

ID IDID

ID ID ID

ID ID

l l l

ll

SYBR GreenI Primer Characteristics

Many of the guidelines for primer design are also

applicable to SYBR Green primer design.

Create amplicon length of 200-300 bp.

Avoid primer-dimers!!!

96

R

Q

RQ

TaqManTM probe

After cleveage

Before cleveage

TaqManTM

5’

5’

3’

3’

d.NTPs


Primers Add Master Mix and Sample

Denaturation

Annealing

Reaction Tube

Taq

l

R Q

Probe

R Q

5’ 3’

TaqManTM

RQ

Extension Step

5’ 3’ 1. Strand DisplacementTaq

Q

R

5’ 3’

Q Taq

R

5’ 2. Cleavage

3. PolymerizationComplete

5’ 3’

TaqR

5’

4. Detection5’ 3’

TaqR

5’

l

R

TaqMan Design characteristics-A

Many of the guidelines for primer design are also

applicable to TaqMan® probe design.

The probe melting temperature in general should be ~10؛C

higher than the forward or reverse primer.

The probe should be as close to the forward OR reverse

primers as possible, within 10 base pairs of the primer

that anneals to the same strand as the probe without

any overlaps

100

TaqMan Design characteristics-B

The amplicon size is usually in the range of 60–150.

Do not put G at the 5’ end of the probe as this will quench

reporter fluorescence.

In general, probes with more G than C bases will often

produce reduced normalized fluorescence values.

101

102

Molecular beacons

Molecular beacons

Reporter

Non-fluorescent Quencher

Amplicon

A

B

C

FRET

Excitation

ANNEALING

FITC

Red 640

P Phosphate

FRET Emission

P

Excitation

Amplicon

FRET Design characteristics

Many of the guidelines for primer design are also applicable

to FRET probe design.

Amplicon should be between 100-200.

Place probes far away from the primer on the same strand.

Length of 25 to 35 nucleotides.

TM 5 to 8°C above primer TM.

No Gs should be located in the target strand between the

annealed probes.

105

The Basic of Real time PCR

Baseline – The baseline phase contains all the amplification that is below the level of

detection of the real time instrument.

Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically

CT – (cycle threshold) the cycle number where the fluorescence passes the threshold

DRn – (Rn-baseline)

NTC – no template control

RT control

Melting Curve

Log

flu

ore

scen

ce (

Rn

)

NTC Controls

NTC’s- Negative Control Rxn with Primers but without Template Examine Amp Plots to determine whether or not you have any amplification products.

The presence of an amplication product is indicative of contamination

Sources of contamination: water, primers, SYBR Green Master Mix, Pipets, etc

RT control

• Genomic DNA contamination

• Primer design (Exon-exon junction)

Melt Curve Analysis

Melt Curve used to Assess Specificity of Rxn Do you have a single amplification peak or multiple amplification peaks?

Single Peak= Single Product

Multiple Peaks= Multiple Products

What is the peak melt temperature and how does this correlate with the expected melt peak temperature?

To further verify that you have amplified correct product can sequence amplification product

Output data: Melt curve*

Relative fluorescence units

Gradual temperature-dependent fluorescencequenching

Rapid decrease in fluorescence caused by denaturation of dsDNA (PCR product)

Negative first derivative offluorescence/temperature

Melt peak (85.5º C)

Identification of multiple PCR products using a melt curve

product 2(Tm 86.9º C)

Under identical solvent conditions, Tm is determined by G/C content and length of dsDNA

primer dimer(Tm 77.0º C) product 1

(Tm 85.5º C)

Reverse Transcription

Total RNA 5 g

random hexamers (50 ng/l) 3 l

10 mM dNTP mix 1 l

DEPC H2O to 10 l

10x RT buffer 2 l

25 mM MgCl2 4 l

0.1 M DTT 2 l

RNAaseOUT 1 l

Incubate the samples at 65C for 5 min and then on ice for at least 1 min.

Add 1 µl (50 units) of SuperScript II RT to each tube, mix and incubate at 25C for 10 min.

Incubate the tubes at 42C for 50 min, heat inactivate at 70C for 15 min, and then chill on ice.

25 l SYBR Green Mix (2x)

0.5 l cDNA

2 l primer pair mix (5 pmol/l each primer)

22.5 l H2O

OR

12.5 l SYBR Green Mix (2x)

0.2 l cDNA

1 l primer pair mix (5 pmol/l each primer)

11.3 l H2O

Tth DNA Polymerase

Platinum® Taq DNA polymerase

GoTaq® Hot Start Polymerase

Hot Start II High-Fidelity DNA Polymerase

95C 10 min, 1 cycle 95 C 15 s -> 60 C 30 s -> 72 C 30 s, 40 cycles

Results interpretation

Following run evaluation

Valid positive and negative control

Specimen has a normal curve

Record the cycle threshold (Ct) values

If a sample has no cycle threshold values (0.00) it is negative

Determine if there are any suspect samples

Weak positives- Ct values >35

Results interpretation

Following run evaluation

Valid positive and negative control

Specimen has a normal curve

Record the cycle threshold (Ct) values

If a sample has no cycle threshold values (0.00) it is negative

Determine if there are any suspect samples

Weak positives- Ct values >35

* Absolute quantification* Relative quantification (relative fold change)

i. Relative standard method ii. Comparative CT (2 -DDCT) method

Types of real-time PCR quantification

118

STANDARD CURVE

METHOD

120

CYCLE NUMBER AMOUNT OF DNA

0 1

1 2

2 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1,024

11 2,048

12 4,096

13 8,192

14 16,384

15 32,768

16 65,536

17 131,072

18 262,144

19 524,288

20 1,048,576

21 2,097,152

22 4,194,304

23 8,388,608

24 16,777,216

25 33,554,432

26 67,108,864

27 134,217,728

28 268,435,456

29 536,870,912

30 1,073,741,824

31 1,400,000,000

32 1,500,000,000

33 1,550,000,000

34 1,580,000,000

121

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBERA

MO

UN

T O

F D

NA

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

CYCLE NUMBER AMOUNT OF DNA

0 1

1 2

2 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1,024

11 2,048

12 4,096

13 8,192

14 16,384

15 32,768

16 65,536

17 131,072

18 262,144

19 524,288

20 1,048,576

21 2,097,152

22 4,194,304

23 8,388,608

24 16,777,216

25 33,554,432

26 67,108,864

27 134,217,728

28 268,435,456

29 536,870,912

30 1,073,741,824

31 1,400,000,000

32 1,500,000,000

33 1,550,000,000

34 1,580,000,000

122

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

123

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

124

SERIES OF 10-FOLD DILUTIONS

threshold

Ct

125

126

127

15SERIES OF 10-FOLD DILUTIONS

threshold

• This method assumes all standards and samples have

approximately equal amplification efficiencies

• More labour-intensive

because of the necessity to create reliable standards for

quantification

include these standards in every PCR

not possible to use DNA as a standard for absolute quantitation of

RNA because there is no control for the efficiency of the reverse

transcription

• Accurate determination of total RNA concentration is particularly

important

quantification by OD measurement faces problem of DNA

contamination or inaccurate results from the spectrophotometer

RNA constituting on average only 50-80% of the purified nucleic

acid

Additional step of DNase removal should be carried out prior to

any RT step

Absolute quantification

Relative standard curve method1. Construct a relative

standard curve

2. Calculate the input amount by entering the following formula in an adjacent cell:= 10^ [cell containing log input amount]

3. Divide the amount of c-myc by the amount of GAPDH to determine the

normalized amount of c-myc (c-mycN).

* Absolute quantification* Relative quantification

(relative fold change vs calibrator)i. Relative standard curve methodii. Comparative CT (2 -DDCT) method

Types of real-time PCR quantification

3. PCR Quantification

Quantification and Normalization

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

• First basic underlying principle: every cycle there is a doubling of product.

• Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities.

• Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene.

• Key formula:

– Quantity = 2 ^ (Cta

– Ctb

)

Housekeeping Gene for Normalization

Housekeeping Gene for Normalization

1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2. b -actin3. Ribosomal RNA (rRNA): 28S, 18S

None of the identified reference for data normalization are ideal.

GAPDH

(Bustin, 2000)

GAPDH concentrations vary• between different individuals (Bustin et al. 1999),• during pregnancy (Cale et al. 1997), • with developmental stage (Puissant et al. 1994, Calvo et al. 1997),• during the cell cycle (Mansur et al. 1993),• apoptosis (Ishitani et al. 1997)• food deprivation (Yamada et al. 1997)*** Inducer• after the addition of the tumour promoter 12-Otetradecanoyl-phorbol-13-acetate (Spanakis

1993),dexamethasone (Oikarinen et al. 1991) and carbon tetrachloride (Goldsworthy et al. 1993).• Insulin stimulates GAPDH transcription (Rolland et al 1995, Barroso et al. 1999) • calcium ionophore A23187 induces GAPDH transcription • Growth hormone (Freyschuss et al. 1994), vitamin D (Desprez et al. 1992), oxidative stress (Ito et

al. 1996), hypoxia (Graven et al. 1994, Zhong & Simons 1999), manganese (Hazell et al. 1999) and the tumour suppressorTP53 (Chen et al. 1999), have all been shown to activate its transcription

• retinoic acid (Barroso et al. 1999) downregulate GAPDH transcription in the gut and in adipocytes, respectively.

*** unregulated in cancer• in rat hepatomas (Chang et al. 1998),• Malignant murine cell lines (Bhatia et al. 1994) and• Human prostate carcinoma (Ripple & Wilding 1995)

Its use as an internal standard is inappropriateIt is a mystery why GAPDH continues to find favor as an internal standard.

4. Housekeeping Gene for Normalization

b-actin concentrations vary widely in response to

• experimental manipulation in human breast epithelial cells (Spanakis 1993)

• in various porcine tissues (Foss et al. 1998) canine myocardium (Carlyle et al. 1996)

• the presence of pseudogenes interferes with the interpretation of results (Dirnhofer et al. 1995, Raff et al. 1997, Mutimer et al. 1998)

• primers commonly used for detecting -actin mRNA amplify DNA (Dakhama et al. 1996).

4. Housekeeping Gene for Normalization

Common normalizing“housekeeping gene”

•Ribosomal RNA (rRNA)

– 28S, 18S

Varying ratios of rRNA to mRNA have been reported (Solanas et al.,2001)

Use random hexamer instead of oligo dT in the RT step

Comparative CT method-I

ABI-7700 User Bulletin #2

Comparative CT method - II

ABI-7700 User Bulletin #2

Relative quantification

Gapdh IL-2 delta delta delta RGE

Cont1 15 27 12 0 1 32

RA1 16 23 7 -5 32

Gapdh IL-2 delta delta delta RGE

Cont1 15 27 12 5 0.03125 32

RA1 16 23 7 0 1

10

Gapdh IL-2 delta delta delta RGE 32

Cont1 15 27 12 2 0.25

RA1 16 23 7 -3 8

0

Gapdh IL-2 delta delta deltaRGE

Cont1 15 27 12 12 0.00024414 1 32

RA1 16 23 7 7 0.0078125 32

targettreated

ref treated

targetcontrol

ref control

av =19.80

av =19.93

av =18.03

av =29.63

D Ct = 9.70

D Ct = -1.7

D Ct = target - ref

D Ct = target - ref

Difference = DCt-DCt= DDCt= (-1.7) -9.70= -11.40

control

experiment

Exercise: By 2 –∆∆CT, fold change=??? 2702

3. PCR Quantification

145

PFAFFL METHOD

– M.W. Pfaffl, Nucleic Acids

Research 2001 29:2002-2007

EFFECTS OF EFFICIENCY

146

147

AFTER 1 CYCLE100% = 2.00x90% = 1.90x80% = 1.80x70% = 1.70x

CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA

100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY

0 1 1 1 1

1 2 2 2 2

2 4 4 3 3

3 8 7 6 5

4 16 13 10 8

5 32 25 19 14

6 64 47 34 24

7 128 89 61 41

8 256 170 110 70

9 512 323 198 119

10 1,024 613 357 202

11 2,048 1,165 643 343

12 4,096 2,213 1,157 583

13 8,192 4,205 2,082 990

14 16,384 7,990 3,748 1,684

15 32,768 15,181 6,747 2,862

16 65,536 28,844 12,144 4,866

17 131,072 54,804 21,859 8,272

18 262,144 104,127 39,346 14,063

19 524,288 197,842 70,824 23,907

20 1,048,576 375,900 127,482 40,642

21 2,097,152 714,209 229,468 69,092

22 4,194,304 1,356,998 413,043 117,456

23 8,388,608 2,578,296 743,477 199,676

24 16,777,216 4,898,763 1,338,259 339,449

25 33,554,432 9,307,650 2,408,866 577,063

26 67,108,864 17,684,534 4,335,959 981,007

27 134,217,728 33,600,615 7,804,726 1,667,711

28 268,435,456 63,841,168 14,048,506 2,835,109

29 536,870,912 121,298,220 25,287,311 4,819,686

30 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

148

AFTER 1 CYCLE100% = 2.00x90% = 1.90x80% = 1.80x70% = 1.70x

AFTER N CYCLES:fold increase = (efficiency)n



0 1 1 1 1

1 2 2 2 2

2 4 4 3 3

3 8 7 6 5

4 16 13 10 8

5 32 25 19 14

6 64 47 34 24

7 128 89 61 41

8 256 170 110 70

9 512 323 198 119

10 1,024 613 357 202

11 2,048 1,165 643 343

12 4,096 2,213 1,157 583

13 8,192 4,205 2,082 990

14 16,384 7,990 3,748 1,684

15 32,768 15,181 6,747 2,862

16 65,536 28,844 12,144 4,866

17 131,072 54,804 21,859 8,272

18 262,144 104,127 39,346 14,063

19 524,288 197,842 70,824 23,907

20 1,048,576 375,900 127,482 40,642

21 2,097,152 714,209 229,468 69,092

22 4,194,304 1,356,998 413,043 117,456

23 8,388,608 2,578,296 743,477 199,676

24 16,777,216 4,898,763 1,338,259 339,449

25 33,554,432 9,307,650 2,408,866 577,063

26 67,108,864 17,684,534 4,335,959 981,007

27 134,217,728 33,600,615 7,804,726 1,667,711

28 268,435,456 63,841,168 14,048,506 2,835,109

29 536,870,912 121,298,220 25,287,311 4,819,686

30 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

149

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF



0 1 1 1 1

1 2 2 2 2

2 4 4 3 3

3 8 7 6 5

4 16 13 10 8

5 32 25 19 14

6 64 47 34 24

7 128 89 61 41

8 256 170 110 70

9 512 323 198 119

10 1,024 613 357 202

11 2,048 1,165 643 343

12 4,096 2,213 1,157 583

13 8,192 4,205 2,082 990

14 16,384 7,990 3,748 1,684

15 32,768 15,181 6,747 2,862

16 65,536 28,844 12,144 4,866

17 131,072 54,804 21,859 8,272

18 262,144 104,127 39,346 14,063

19 524,288 197,842 70,824 23,907

20 1,048,576 375,900 127,482 40,642

21 2,097,152 714,209 229,468 69,092

22 4,194,304 1,356,998 413,043 117,456

23 8,388,608 2,578,296 743,477 199,676

24 16,777,216 4,898,763 1,338,259 339,449

25 33,554,432 9,307,650 2,408,866 577,063

26 67,108,864 17,684,534 4,335,959 981,007

27 134,217,728 33,600,615 7,804,726 1,667,711

28 268,435,456 63,841,168 14,048,506 2,835,109

29 536,870,912 121,298,220 25,287,311 4,819,686

30 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

150

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF

151

IL1-b con

IL1-b vit

RPLP0 vit

RPLP0 con

152

IL1-b con

IL1-b vit

AFTER N CYCLES: change = (efficiency)n

AFTER N CYCLES: ratio vit/con = (1.93)29.63-18.03 =1.9311.60 = 2053

av =18.03 av =29.63

IL1-beta

153

ratio = change in IL1-B = 2053/1.08 = 1901change in RPLP0

ratio = (Etarget )DCt target (control-treated)

(Eref )DCt ref (control-treated)

154

DDCt EFFICIENCY METHOD

APPROXIMATION METHOD

155

IL1-b con

IL1-b vit

RPLP0 vit

RPLP0 con

156

IL1-b vit

RPLP0 vit

IL1-b con

RPLP0 con

av =19.80

av =19.93

av =18.03

av =29.63

D Ct = 9.70

D Ct = -1.7

D Ct = target - ref

D Ct = target - ref

Difference = DCt-DCt= DDCt = 9.70-(-1.7)= 11.40

control

experiment

DDCt = 11.40 for IL1-beta

• 2 DDCt variant: assumes efficiency is 100% Fold change = 211.40 = 2702

• But our efficiency for IL1-beta is 93%

– Fold change = 1.9311.40 = 1800

• Pfaffl equation corrected for RPLP0 efficiency

– Fold change = 1901

157

• assumes

– minimal correction for the standard gene, or

– that standard and target have similar efficiencies

• 2 DDCt variant assumes efficiencies are both 100%

• approximation method, but need to validate that assumptions are reasonably correct - do dilution curves to check DCts don’t change

• The only extra information needed for the Pfaffl method is the reference gene efficiency, this is probably no more work than validating the approximation method

158

DDCt EFFICIENCY METHOD

real-time polymerase chain reaction

Health & Medicine

dna polymerase activity

dna presentless dna

agarose gel

native taq dna polymerase

green gotaq reaction

buffer troubleshootingbuffer

primers buffer considerations

trisbased buffer