MBG-487 Real-Time Quantitative RT-PCR. Agarose EtBr Gel.

MBG-487

Real-Time Quantitative RT-

PCR

Agarose EtBr Gel

PCR Phases

PCR Phases in Linear view

Real-Time Vs Traditional PCR

• detection of PCR amplification during the early phases of the reaction.

• detection of PCR amplification at the final phase or end-

point of the PCR reaction by agarose gels.

• Real-Time PCR can detect as little as a two-fold change! Agarose Gel resolution is very poor, about 10 fold.

• Real-Time PCR detects the accumulation of amplicon during the reaction

THE PROBLEM

• NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION

• SMALL AMOUNTS OF mRNA– LASER CAPTURE– SMALL AMOUNTS OF TISSUE– PRIMARY CELLS– PRECIOUS REAGENTS

Software-based analysis

•Data acquisition

•Fluorescence in each well at all cycles.

•Software-based curve fit of fluorescence vs cycle number

•Threshold

•Fluorescence level that is significantly greater than the baseline.

•Automatically determined/User controlled

•CT (Cycle threshold)

•Cycle at which fluorescence for a given sample reaches the threshold.

•CT correlates, inversely, with the starting concentration of the target.

• Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly, and is a preferable alternative to other forms of quantitative RT-PCR that detect the amount of final amplified product at the end-point

•By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template

•The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed

•Real-time PCR also offers a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR).

• There are two main fluorescence-monitoring systems for DNA amplification

(1) DNA-binding agents (SYBR GREEN)

(2) Taqman probes

SYBR Green DyeSYBR Green chemistry is an alternate method used to perform real-timePCR analysis. SYBR Green is a dye that binds the Minor Groove ofdouble stranded DNA. When SYBR Green dye binds to double strandedDNA, the intensity of the fluorescent emissions increases. As moredouble stranded amplicons are produced, SYBR Green dye signal willincrease. SYBR Green dye will bind to any double stranded DNAMolecule.

SYBR Green is very sensitive; it is 25 times more sensitive than ethidium bromide, another commonly used dye for visualizing DNA. The high affinity of SYBR Green for double stranded DNA makes it useful for detecting samples of DNA with low copy number. It preferentially binds double stranded DNA, but it can also bind single stranded DNA with reduced fluorescence. It is frequently used in real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide)

SYBR GREEN

SYBR® green I

•Non specific

Intercalation, binding structure dependent, not sequence dependent

•SYBR green fluorescence

•Quantification and characterisation via melt curves

•Careful primer design necessary to avoid primer dimers

IT IS NOT SPECIFIC BUT IT IS SENSITIVE!!!

SYBR Green Dye Assay

Taqman ® probes

• Specific

• Binds only to specific target

• Taqman probe binds first, then primers during annaeling phase, elongation by Taq polymerase, 5’Exonuclease activity of Taq polymerase cleaves fluorophore off probe ⇑fluorescence

• Probe system with highest signal

• Qunatification and allelic discrimination (SNP detection)

• Easy design using PrimerExpress™or BeaconDesigner

IT IS SPECIFIC BUT NOT SENSITIVE!!!

FRET (Fluorescent Resonance Energy Transfer)FRET or Florescent Resonance Energy Transfer technology is utilized inthe 5’ nuclease assay. The principle is that when a high-energy dye is inclose proximity to a low-energy dye, there will be a transfer of energyfrom high to low.

The 5’ Nuclease Assay

In the 5’ nuclease assay, an oligonucleotide called a TaqMan Probe is

added to the PCR reagent master mix. The probe is designed to anneal

to a specific sequence of template between the forward and reverse

primers. The probe sits in the path of the enzyme as it starts to copy DNA

or cDNA. When the enzyme reaches the annealed probe the 5’ exonuclease

activity of the enzyme cleaves the probe.

The 5' Nuclease Assay

Polymerase collideswith TaqMan Probe

Cleavage of the TaqMan Probe

The TaqMan Probe is designed with a high-energy dye termed a Reporter at the 5' end, and

a low-energy molecule termed a Quencher at the 3' end. When this probe is intact and

excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a

result

of the close proximity of the dyes.

When the probe is cleaved by the 5’ nuclease activity of the enzyme, the distance between the

Reporter and the Quencher increases causing the transfer of energy to stop. The fluorescent

emissions of the reporter increase and the quencher decrease.

Increased florescence activity due to the cleaved probe

The point at which the fluorescence crosses the threshold is called the Ct. The threshold cycle (Ct) is when the system begins to detect the increase in the fluorescent signal associated with an exponential growth of PCR product during the log-linear phase.

A Ct value of 40 or higher means no amplification and this value cannot be included in the calculations

All PCR products for a particular primer pair should have the same

melting temperature - unless there is contamination, mispriming1,

primer-dimer2 artifacts, or some other problem

• Primer Design• Amplification Efficiency

For the highest efficiency in real-time RT-PCR using SYBR Green, targets should ideally be 100–200 bp in length.

• Reference Gene Selection

CONSIDERATIONS IN REAL TIME RT-PCR

Importance of Primers in PCR

• specific

• high efficiency

• no primer-dimers

• Ideally should not give a DNA signal– cross exon/exon boundary

AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x

Efficency Calculation

• Eff can be calculated by the formula:

Eff = 10(-1/slope) – 1

• The efficiency of the PCR should be

90 - 100% (– 3.6 > slope > – 3.1)

10^-(1/-3.4)= 1.96

(1.96-1)*100= 96% efficient


• Choosing a proper internal control (housekeeping) gene for normalization.

• The internal control gene(s) should not vary in the tissues or cells under investigation.

• Minimal number of most stable genes should be used.

• For the averaging of the selected genes geometric mean is more accurate than arithmetic.

SELECTION OF

QUANTIFICATION METHOD

(DATA ANALYSIS METHOD)

QUANTITATION OF mRNA LEVELS USING REAL TIME PCR

• STANDARD CURVE METHOD

• Delta-Delta CT METHOD (An approximation method)

• PFAFFL METHOD

STANDARD CURVE METHOD

Dilution curve target gene

‘copy number’ target gene experimental

‘copy number’ target gene control

fold change in target gene=copy number experimentalcopy number control




• PFAFFL METHOD

2-(Ct)

Ct:[(Cttumor-Cthousekeeping)-(Ctnormal-Cthousekeeping)]




• PFAFFL METHOD

Efficiency Method

target gene

internal control geneactin, GAPDH, RPLP0 etc

Ratio target gene in experimental/control = fold change in target gene fold change in reference gene

control expt

Corrected fold increase = 10/2 = 5

REAL TIME PCR

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MBG-487 Real-Time Quantitative RT-PCR. Agarose EtBr Gel.

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