PCR quantitativeen temps réel

Lydie Pradel

PCR

PCR semi-quantitative25 cycles

Sybr Green Fluorogenic 5’Nuclease Assay

Binds dsDNA 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 Probe Sybr Green

Specificityprimer binding Primer bindingProbe hybridizationPCR conditions PCR conditions

FlexibilityMultiplex easy, only primers neededSNP detection

OptimizationCkeck primer dimer formation

Thermal Cycling Protocol (Applied Biosystem)

95°C 10’ Activation of AmpliTaq Gold Polymerase95°C 15’’ Denaturation60°C 1’ 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

RnFAM dye

ROX dye

Rn

Sample 1 Sample 2

Rn= Reporter/Passive reference

Fluo

recs

ence

Fluo

recs

ence

From fluorescence to results

105 104 103 104 103

Primer specificity: efficiency

If slope= -3,32 efficiency becomes 1

Quantification

Absolute quantificationStandard 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

timet=0 t=12 t=24 t=48

Total RNA Total RNA Total RNA Total RNA

cDNA cDNA cDNA 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 ?

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= 0 t= 12h

Threshold Threshold

Ct = Ct target gene – Ct Endogenous gene

Ctt=0 35-15= 20 Ctt=12 30-15= 15

Ct = Ct Sample - Ct Calibrator

Ct 15-20= -5

2-Ct

2^-(-5)= 32

Relative quantification of the 4 samples

50

40

30

20

0

10

Samples

X-f

old

exp

ress

ion

t = 0

t = 12 h

t = 24 h

t = 48 h

Calibratort=0

1

32 35

4