1. Real Time PCR =
Quantitative PCR

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

3. Measure/ compare the quantity of (DNA) molecules of interest
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. Sequence specific probe
2 technical alternatives for precise detection of PCR product :
Sequence specific probe
Sybr green

5. Sequence Specific
Probe =
Taqman probe R Q

6. 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 R1 R2
transfer
excitation
emission

7. Sybr green

8. 2 products of amplification
Additional control for sybr green PCR = dissociation curve
So you can design new (more specific) primers or use a taqman probe to increase the specificity of your reaction. Indeed, a non-specific reaction is very often due to a bad primers design.
1 product of amplification
2 products of amplification

9. Measure/ compare the quantity of (DNA) molecules of interest
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
plateau
Only during exponential phase, the fluorescent signal emitted by the probe (or Sybr green) reflects the increasing amount of PCR product
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. Measure/ compare the quantity of (DNA) molecules of interest
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 have PCR reaction 100% efficient = to have good amplicons
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
The most delicate step of a Q-PCR reaction is the design of the primers and probe that have to match different parameters to give the most efficient PCR reaction. These parameters depend on the unusual cycle of reaction that have very short steps but above all where annealing and elongation take place at the step and at 60°C

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
The most delicate step of a Q-PCR reaction is the design of the primers and probe that have to match different parameters to give the most efficient PCR reaction. These parameters depend on the unusual cycle of reaction that have very short steps but above all where annealing and elongation take place at the step and at 60°C
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
intron 1
intron 1
intron 1
intron 1
ex 2
ex 2
ex 2
ex 2

16. Additional parameters:
- on cDNA: the amplicon overlaps an exon-exon junction to prevent amplification of genomic DNA
Additional parameters:
- 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. Ct: number of cycles required to reach the threshold
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 =
Efficiency = 1.98 ( %)

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

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

29. Expression analysis Experimental design
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 work very precisely during PCR assembly
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 work very precisely during PCR assembly
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. DDCt Method Normalization step DCt Target gene House keeping gene
Sample 1
Sample 2
House keeping gene
Sample 1
Sample 2 26 27 27 28
DCt
Sample 1 DCt = 26-27= -1
Sample 2 DCt = 27-28= -1

33. Sample 2 vs Sample = 2-DDCt = 1
DDCt Method
Enrichment factor
Target gene
Sample 1
Sample 2
House keeping gene
Sample 1
Sample 2 26 27 27 28
DCt
DDCt
Sample 1 DCt = 26-27= -1
Sample 2 vs Sample 1 = 0
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. Goal: to determine the exact number of template in the sample
Absolute quantification Ct
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