Real-Time Quantitative RT-PCR MBG-487 Real-Time Quantitative RT-PCR

Agarose EtBr Gel

PCR Phases

PCR Phases in Linear view

But of course, the reaction can’t go on forever, and it eventually tails off and reaches a plateau phase, as shown by the figures in red. If we plot these figures in the standard fashion (top), we cannot detect the amplification in the earlier cycles because the changes do not show up on this scale. Eventually you see the last few cycles of the linear phase (pink) as they rise above baseline and then the non-linear or plateau phase (red) - in real life this starts somewhat earlier than shown here. If we plot the amount of DNA on a logarithmic scale, we can see the small differences at earlier cycles - in real time PCR we use both types of graph to examine the data. Note that there is a straight line relationship between the amount of DNA and cycle number when you look on a logarithmic scale - because PCR amplification is a logarithmic reaction.

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 Real time PCR was developed because of the need to quantitate differences in mRNA expression. PCR methods are particularly valuable when amounts of RNA are low since the fact that they involve an amplification step means they are more sensitive.

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 DNA-binding agents (SYBR GREEN) (2) Taqman probes

SYBR Green Dye SYBR Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR Green is a dye that binds the Minor Groove of double stranded DNA. When SYBR Green dye binds to double stranded DNA, the intensity of the fluorescent emissions increases. As more double stranded amplicons are produced, SYBR Green dye signal will increase. SYBR Green dye will bind to any double stranded DNA Molecule.

SYBR GREEN 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) In this presentation, we will be using Sybr green to monitor DNA synthesis. Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does, which is why we use Sybr Green rather than ethidium bromide; we also use Sybr green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of single-stranded DNA is much higher that the ratio for ethidium bromide). Other methods can also be used to detect the product during real-time PCR, but will not be discussed here. However, many of the principles discussed below apply to any real-time PCR reaction.

•SYBR green fluorescence 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

IT IS SPECIFIC BUT NOT SENSITIVE!!! 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 in the 5’ nuclease assay. The principle is that when a high-energy dye is in close proximity to a low-energy dye, there will be a transfer of energy from 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 collides with 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

CONSIDERATIONS IN REAL TIME RT-PCR 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

Importance of Primers in PCR specific high efficiency no primer-dimers Ideally should not give a DNA signal cross exon/exon boundary

CONSIDERATIONS IN REAL TIME RT-PCR 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

AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x Here is a series of calculations as to how much the DNA will be amplified if you have different efficiencies. For 100% efficiency, there will be a doubling at each cycle, for 90% the amount of DNA will increase from 1 to 1.9 , so the factor is 1.9 for each cycle, and similarly for 80% and 70% it will be 1.8 and 1.7. Notice that a small different in efficiency makes a lot of difference in the amount of final product. Each 10% lowering results in less than 25% of the previous column after 30 cycles.

Since the lines diverge at higher thresholds, lower thresholds will minimize the error due to small changes in efficiency. If a reaction has an inhibitor of PCR in it which reduces the efficiency, the slope will be different from unaffected reactions when you look at the results using the logarithmic scale. Thus if you do triplicate reactions and one has a bad slope, you should drop that well from the analysis.

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

CONSIDERATIONS IN REAL TIME RT-PCR 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 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 There are several methods to quantitate alterations in mRNA levels using real time PCR, let’s look at the standard curve method first.

fold change in target gene= copy number experimental ‘copy number’ target gene control Dilution curve target gene ‘copy number’ target gene experimental fold change in target gene= copy number experimental copy number control You tell the software which dilution curve you want to use, and which unknowns you want it to quantitate using that curve. You don’t actually give it a real copy number - just start at some at some arbitrary number. The machine will report the copy number or amount of DNA in each of your unknown samples and even average this for you. If you take the average of the copy number of the target gene in the experimental sample, divide it by the average copy number in the control sample,this will give the fold change in the target gene. You have now calculated the upper value in the ‘Northern’ formula we derived earlier on (slide 4). Note the excellent fit of the standard curve data to a straight line - a perfect fit would have a correlation coefficient of 1.000 and here the correlation coefficient is 0.999.

QUANTITATION OF mRNA LEVELS USING REAL TIME PCR STANDARD CURVE METHOD Delta-Delta CT METHOD   (An approximation method) PFAFFL METHOD 2-(Ct) Ct:[(Cttumor-Cthousekeeping)-(Ctnormal-Cthousekeeping)]

QUANTITATION OF mRNA LEVELS USING REAL TIME PCR STANDARD CURVE METHOD Delta-Delta CT METHOD   (An approximation method) PFAFFL METHOD

Efficiency Method

10X 2X Corrected fold increase = 10/2 = 5 target gene control expt target gene 10X internal control gene actin, GAPDH, RPLP0 etc 2X One question is how one uses real-time PCR to quantitate the amounts of DNA or cDNA. The first two calculation methods for real-time PCR results that we are going to focus on are equivalent to the calculations that one usually does when one does a Northern. This slide shows a virtual Northern with two lanes, one with RNA from control cells, the other with RNA from the experimental sample (eg drug treated cells). For the sake of argument, let’s say that there is 10x the amount of signal in the experimental sample compared to the control sample for the target gene. This could mean expression of the gene has increased 10-fold, or it could mean that there is 10x as much RNA in the expt lane. To check for this one usally does a so-called ‘loading control’ in which the blot is probed for expression of a gene which does not change (e.g. actin, GAPDH, cyclophilin, RPLP0 mRNAs; ribosomaL RNA). In this case, let’s say that the loading control shows that there is twice as much RNA in the expt lane. Thus the real change in the target gene is 10/2 =5 fold. We can express this in a more general fashion: ratio target gene (experimental/control) = fold change in target gene (expt/control) fold change in reference gene (expt/control) Corrected fold increase = 10/2 = 5 Ratio target gene in experimental/control = fold change in target gene fold change in reference gene

REAL TIME PCR Real time PCR is a kinetic approach, where you look at the reaction in the early stages while it is still linear. There are many real time machines available. This is the one we use (the BioRad Icycler IQ real time PCR instrument). The lid slides back and then we put samples in a 96-well plate format inside, so one can look at a lot of samples simultaneously. The machine contains a sensitive camera which monitors the fluorescence in each well of the 96-well plate at frequent intervals during the PCR reaction. In our case, as DNA is synthesized, more SYBR green will bind and the fluorescence will increase. www.biorad.com