9 - Real time PCR

Comparing qPCR vs PCR

• qPCR - monitors amplification in real time using flourescence, no need for electrophoresis, quantifies gene expression, measures how much target DNA is present in sample during the early exponential phase

• End point PCR - amplification checked after completion by electrophoresis, no correlation between amount of starting DNA and final product due to plateu phase variability caused by reagent depletion or competition

qPRC principle

• Amplification is proportional to starting DNA at the early exponential phase

• SYBR green - dye that flouresces when bound to dsDNA

• TaqMan probe - 5’ reporter flourophore (FAM) and 3’ quencher that blocks flourescense until probe is cleaved

• Probe binds to DNA, during elongation taq polymerase 5’-3’ exonuclease activity degrades probe, reporter separates from quencher, flourescence emitted

Pros and cons of SYBR green and TaqMan

• SYBR green - low cost, simple, binds non specifically to any dsDNA, no multiplexing, requires melt curve to compare target

• TaqMan - highly specific to target due to custom sequence, expensive due to custom probes, multiplexing (different flourophores), no melt curve

qPCR machine

• Thermal cycler

• Excitation filter - optimises wavelength to excite flourophores

• Emission filter - captures flourescence emitted by dyes or probes

• Amplification plot - flourescence Y axis vs PCR cycles X axis

Applications of qPCR

• Gene expression analysis - measures mRNA levels

• Pathogen detection - quantify viral / bacterial DNA

• Genotyping - detect SNPs / mutations

• GMO testing to verify transgenic DNA

Threshold cycle (ct)

• Cycle number at which flourescense surpasses background noise (detectable amplification)

• Lowe ct indicated higher starting DNA, ct of 20 vs 30 means 1000x more DNA

• Baseline - background flourescence (3-15 cycles)

• Treshold - set 10 standard deviations above baseline, but not always

Absolute quantification

• Measures exact copy number by using a standard curve

• The log of known DNA values in plotted against their ct values, the ideal slope is -3.32 (100% efficiency)

• 3.32 change is ct implies a 10% dilution

Relative quantification (RT-qPCR)

• Measures fold changes in gene expression e.g. drug treated vs control

• Reverse transcriptase to convert mRNA to cDNA

• Housekeeping gene (endogenous control) that always has the same expresion (GAPDH) will correct errors like pipetting

• You will get two ct values, one for housekeeping gene and one for sample, compare the fold change

• Compare the change to the calibrator sample (e.g. cells with normal expression) (ΔΔCt)

Example of calculating fold change

• Ct target gene = 22

• Ct housekeeping gene = 20

• Change = 2

• Subtract change of test sample from change of calibrator e.g. 2-5 = -3

• 2^3 = 8x more expressed(change sign)

• 2^-2 = 0.25 = 4x less expressed

ROX dye, melt curve analysis and no template control

• ROX - passive reference flourescent dye that normalises flourescence across samples, corrects pipetting errors or machine variability

• MCA - checks amplicon purity by measuring Tm, single peak means specific amplification, multiple means primer dimers or contamination

• NTC - negative control to detect contamination (ct over 35), amplification in this well invalidates results

Rn value

• Normalised reporter flourescence

• Divide reporter flourescence by ROX flourescence value