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PCR quantitative en temps réel

PCR quantitative en temps réel. Lydie Pradel. PCR. PCR semi-quantitative 25 cycles. Sybr Green. Fluorogenic 5’Nuclease Assay. Binds ds DNA. Use Taqman probe. Sybr Green fluoresces upon binding to double stranded PCR product

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PCR quantitative en temps réel

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  1. PCR quantitative en temps réel Lydie Pradel

  2. PCR

  3. PCR semi-quantitative 25 cycles

  4. Sybr Green Fluorogenic 5’Nuclease Assay Binds dsDNA Use Taqman probe

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

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

  7. Non specific amplification (genomic DNA and RT-qPCR)

  8. 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

  9. TaqMan Probe Sybr Green Specificity primer binding Primer binding Probe hybridization PCR conditions PCR conditions Flexibility Multiplex easy, only primers needed SNP detection Optimization Ckeck primer dimer formation

  10. Thermal Cycling Protocol (Applied Biosystem) 95°C 10’ Activation of AmpliTaq Gold Polymerase 95°C 15’’ Denaturation 60°C 1’ Annealing/Extension

  11. Passive reference ROX dye 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. ROX dye normalizes for non-PCR related fluorescence variation Sample 1 Sample 2 FAM dye Rn Fluorecsence Fluorecsence FAM dye ROX dye Rn ROX dye Reporter/Passive reference Rn=

  12. From fluorescence to results 104 103 105 104 103

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

  14. 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

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

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

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

  18. What if we added the double amount of cDNA ? DRn Target gene Endogenous control DCt=23-13=10 Ct=14 Ct=24 Cycles Ct=13 Ct=23

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

  20. Comparative Ct Method calculation Steps Step 1: Normalisation to endogenous control Ct targetgene – Ct Endogenousgene =DCt (do both for calibrator and sample) Step 2: Normalization to calibratorsample DCtSample - DCtCalibrator = DDCt Step3: Use the formula 2-DDCt

  21. t= 0 t= 12h DRn DRn Threshold Threshold TG TG EC EC DCt = Ct target gene – Ct Endogenous gene t= 0 t= 12h DCtt=0 35-15= 20 DCtt=12 30-15= 15 DDCt = DCt Sample - DCt Calibrator Ct=30 Ct=15 Ct=35 Ct=15 Cycles Cycles DDCt 15-20= -5 2-DDCt 2^-(-5)= 32

  22. Relative quantification of the 4 samples t = 0 t = 12 h 50 t = 24 h t = 48 h 40 35 32 30 X-fold expression 20 10 4 1 0 Samples Calibrator t=0

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