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
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