PCR quantitative en temps réel Lydie Pradel

PCR

PCR semi-quantitative 25 cycles

Sybr GreenFluorogenic 5’Nuclease Assay Binds ds DNA Use Taqman probe

Sybr Green fluoresces upon binding to double stranded PCR product Emitted Fluorescence is proportional to amount of amplified product detected in every sample

Specificity check of Sybr Green Gel or melting curve analysis (Real-time PCR system) Sharp, single peak indicates specific amplification

Non specific amplification (genomic DNA and RT-qPCR)

Signal generation with TaqMan Probe Uses2 principles: - FRET technology - 5’-Nuclease activity of the Taq polymerase PCR specificity (primers) Hybridization specificity (probe) Dyes: FAM, VIC, TAMRA

TaqMan ProbeSybr Green Specificity primer bindingPrimer binding Probe hybridizationPCR conditions Flexibility Multiplexeasy, only primers needed SNP detection Optimization Ckeck primer dimer formation

Thermal Cycling Protocol (Applied Biosystem) 95°C10’Activation of AmpliTaq Gold Polymerase 95°C15’’Denaturation 60°C1’Annealing/Extension

This inert dye, whose fluorescence does not change during the reaction, may be added to quantitative, real-time PCR reactions to normalize the well-to-well differences that may occur due to artifacts such as pipetting errors or instrument limitations. Passive reference ROX dye ROX dye normalizes for non-PCR related fluorescence variation FAM dye ROX dye Rn FAM dye ROX dye Rn Sample 1Sample 2 Rn= Reporter/Passive reference Fluorecsence

From fluorescence to results

Primer specificity: efficiency If slope= -3,32 efficiency becomes 1

Quantification Absolute quantification Standard curve Relative quantification Relative increase or decrease No standard curve Calculation of results by comparison of Ct value « comparative Ct method » Definition of - Endogenous Control - Calibrator

Endogenous Control (EC) - Amount of cDNA per well - Constant expression level in all samples - EC normalizes for - RNA input measurement errors - RT efficiency variations Ex: Actine, GAPDH …

Calibrator: an example using four samples time t=0t=12 t=24 t=48 Total RNA cDNA Calibrator

Comparison of Target Gene and Endogenous Control  Ct=24-14=10 Ct=14 Ct=24  Rn Cycles Target gene Endogenous control What if we added the double amount of cDNA ?

Ct=14 Ct=24  Ct=23-13=10  Rn Cycles Target gene Endogenous control Ct=13 Ct=23

Ct=15 Ct=35  Rn Cycles EC TG Comparative Ct method: an example using the four samples Ct=15 Ct=30  Rn Cycles EC TG Ct=9 Ct=24  Rn Cycles EC TG Ct=14 Ct=34  Rn Cycles EC TG t=0 t=12h t=24h t=48h

Comparative Ct Method calculation Steps Step 1: Normalisation to endogenous control Ct target gene – Ct Endogenous gene =  Ct (do both for calibrator and sample) Step 2: Normalization to calibrator sample  Ct Sample -  Ct Calibrator =  Ct Step3: Use the formula 2 -  Ct

Ct=15 Ct=35  Rn Cycles EC TG Ct=15 Ct=30  Rn Cycles EC TG t= 0 t= 12h t= 0t= 12h Threshold  Ct = Ct target gene – Ct Endogenous gene  Ct t= = 20  Ct t= = 15  Ct =  Ct Sample -  Ct Calibrator  Ct 15-20=  Ct 2^-(-5)= 32

Relative quantification of the 4 samples Samples X-fold expression t = 0 t = 12 h t = 24 h t = 48 h Calibrator t=