Real Time PCR = Quantitative PCR

1. Real Time PCR = Quantitative PCR

2. on genomic DNA= measure the copy number of a genomic target sequence on cDNA = compare transcription level of a target gene between different samples (RT-PCR) Goal of Q-PCR: Measure / compare the quantity of (DNA) molecules of interest by PCR

3. Goal of Q-PCR: Measure/ compare the quantity of (DNA) molecules of interest by PCR • Find a way to quantify very precisely the quantity of PCR products • To be sure to compare PCR products during the exponential phase to have a direct proportionality between the signal and the target quantity = 2 times more product / cycle

4. 2 technical alternatives for precise detection of PCR product : Sequence specific probe Sybr green

5. R R Q Q Q Q R R Sequence Specific Probe = Taqman probe

6. excitation transfer R1 R2 Other kinds of probes MGB (Minor Group Binding) probes: same as Taqman probe (with a R and Q) but with a MGB group added at the 5’end of the probe in addition to R, to increase the binding affinity for DNA (increase of the probe Tm) LNA (Locked Nucleic Acid) probe: same as Taqman probe but with modified nucleotides having high affinity for DNA Molecular Beacons: same as Taqman probe but designed to have complementary ends. When the probe is not bound to the target, the 2 ends anneal together and form a stem-loop structure that bring closer the R and Q (--> reduction of the background). FRET (Fluorescence Resonance Energy Transfer) probes: 2 labeled probes that bind to the PCR product in a head-to-tail fashion. When the probes bind to the template, their fluorophores come into close proximity, allowing energy transfer from a donor to an acceptor fluorophore (FRET probes are not cleaved during reaction) Q R emission

7. Sybr green

8. Additional control for sybr green PCR = dissociation curve 1 product of amplification 2 products of amplification

9. Goal of Q-PCR: Measure/ compare the quantity of (DNA) molecules of interest by PCR • Find a way to quantify very precisely the quantity of PCR products • To be sure to compare PCR products during the exponential phase to have a direct proportionality between the signal and the target quantity = 2 times more product / cycle

10. Real time PCR Exponential phase Only during exponential phase, the fluorescent signal emitted by the probe (or Sybr green) reflects the increasing amount of PCR product plateau To be sure to compare signal, from sample to sample, during exponential phase, we measure the signal at each cycle and choose, a posteriori, the optimal cycle number for comparison

11. Goal of Q-PCR: Measure/ compare the quantity of (DNA) molecules of interest by PCR • Find a way to quantify very precisely the quantity of PCR products • To be sure to compare PCR products during the exponential phase to have a direct proportionality between the signal and the target quantity = 2 times more product / cycle PCR 100% efficient

12. To do Q-PCR you need: To have PCR reaction 100% efficient = to have good amplicons

13. On way to have a PCR reaction 100 % efficient is to have PCR cycles as short as possible

14. Design of primers and Taqman probe -Amplicon length: as short as possible (50 to 80n is ideal. Up to 100n for taqman assays is Ok) -Fix one Tm for all the primers = 58°C < Tm < 60°C -Primers lenght: around 20 n (from 15 to 30 is Ok) -Taqman probe Tm must be 10°C greater that PCR primers should not begin with G We use the Primer Express software (Applied Biosystems) for the design of Taqman and Sybr green amplicons

15. Additional parameters: - On cDNA: the amplicon overlapping an exon-exon junction to prevent amplification of genomic DNA cDNA ex 1 ex 1 ex 1 ex 1 ex 2 ex 2 ex 2 ex 2 ex 3 ex 3 ex 3 genomic DNA genomic DNA genomic DNA genomic DNA ex 1 ex 1 ex 1 ex 1 ex 2 ex 2 ex 2 ex 2 intron 1 intron 1 intron 1 intron 1

16. Additional parameters: - on cDNA: the amplicon overlaps an exon-exon junction to prevent amplification of genomic DNA - design in 3’ of the cDNA when the RT is primed with oligo d(T) primer - check by blast if the amplicon selected shows homolgies to other molecules present in the reaction mixture (other mRNA - genomic DNA…)

17. How to test PCR reaction efficiency? by serial dilution of DNA and checking of proportionality between signal and performed dilutions

18. Amplification curves Threshold Ct Threshold: arbitrary fixed level of fluoresence (somewhere in the exponential phase of amplification) Ct: number of cycles required to reach the threshold

19. Cp for systems that use the « second derivative Maximum method » This method identifies the point where the fluorescence curve turns sharply upward the cycle where the second derivative is at its maximum is in the middle of the exponential phase

20. Ct or Cp or Cq These Cq reflects the quantity of target of interest and can be compared between samples

21. Fold dilution 1 X 4 X What is a good amplicon? 16 X 64 X The efficiency of the amplification is calculated by establishing a standard curve (Ct= f(quantity)) When the slope of the standard curve is -3.33, the efficiency = 2 ( 100%) (10-1/slope) = efficiency Slope = -3.366 Efficiency = 1.98 ( 99 %)

22. To do Q-PCR you need: To have good amplicons To have good samples

23. Classical experimental design: To compare the quantity of target between 2 different conditions Ex: mutant vs wt

24. Classical experimental design: To compare the quantity of target between 2 different conditions Ex: mutant vs wt Relative quantification

25. Classical experimental design: To compare the quantity of target between 2 different conditions Ex: mutant vs wt Genomic quantification Expression analysis

26. Experimental design Genomic quantification • To quantify the number of transgene insertions • To select cells that have lost 1 copy of the gene of interest • CNV • Chromatin immuno-precipitation analysis

27. Experimental design Genomic quantification • Clean DNA • need to have several lines of reference (with known number of copies)

28. Experimental design Expression analysis • RNA of good quality (checked with the bioanalyzer - RIN > 7)

29. Experimental design Expression analysis • RNA of good quality (checked with the bioanalyzer - RIN > 7) • Do sample replicates: at least 3 biological replicates + 3 technical replicates (=PCR replicates) • Be as homogenous as possible • ex: cell culture conditions have to be very standardized (use the same medium…) • ex: always compare animals of same age, same parents, same sex… • do paired comparisons if needed

30. To do Q-PCR you need: To have good amplicons To have good samples To work very precisely during PCR assembly - do as much as Master Mix as possible … - liquid handling robots - PCR machines for 384 well plates Rq: if you can’t analyze all your PCR reactions on the same plate, separate between genes, not between samples (for the same gene) - otherwise, duplicate some samples.

31. To do Q-PCR you need: To have good amplicons To have good samples To work very precisely during PCR assembly To have a good method of analysis (normalization method)

32. House keeping gene Sample 1 Sample 1 Sample 2 Sample 2 DDCt Method Normalization step Target gene 26 27 27 28 DCt Sample 1 DCt = 26-27= -1 Sample 2 DCt = 27-28= -1

33. House keeping gene Sample 1 Sample 1 Sample 2 Sample 2 DDCt Method Enrichment factor Target gene 26 27 27 28 DCt DDCt Sample 2 vs Sample 1 = 0 Sample 1 DCt = 26-27= -1 Enrichment factor Sample 2 DCt = 27-28= -1 Sample 2 vs Sample = 2-DDCt = 1

34. Disavantages of the method: • imposes that normalization and target genes efficiencies are equal to 2 • takes in account only 1 house keeping gene

35. GeNorm method (Genome Biology, Vandesompele et al., 2002) • Advanges: • the real amplification efficiency is taken in account • several house keeping genes (HKG) can be used for the normalization • proposes an algorythm that selects the most stable HKG

36. For each new sample conditions, we test at least 4 HKG (6 is best), and we keep the most stable ones according to GeNorm (at least 3)

37. Real-time PCR as validating method for microarrays • ideal method to validate candidates identified by microarrays experiment • -furthermore, Q-PCR is more sensitive than microarrays

38. Ct Absolute quantification Goal: to determine the exact number of template in the sample -synthesis of a external reference = - target synthesized in vitro -determine the exact quantity of synthetized molecules (by spectrophotometry) -do serial dilutions of the reference -(RT)-PCR on the serial dilution of the reference -draw a standard curve for the reference with the exact quantity on the X axis and the Ct on the Y axis Ct -report the Ct obtained for the sample on the standard curve exact quantity