1. Good qPCR The Necessary and the Reasonable

2. 1993 Nobel Prize in Chemistry
PCR origins
1993 Nobel Prize in Chemistry

3. The Polymerase Chain Reaction
Nucleotides
Thermostable DNA polymerase
Forward primer
Reverse primer
Target sequence
Denaturation (~95°C)
Annealing (~60°C)
Extension (72°C)
Cycle 0
Cycle 1
Cycle 2
Cycle 3
Cycle 4
20 = 1 copy
21 = 2 copies
22 = 4 copies
23 = 8 copies
24 = 16 copies

4. End-point analysis is not a viable quantification strategy
adapted from Breljak er al., Food Technol Biotechnol., 2005

5. quantitative PCR (qPCR)
Live tracking of the amplification process allows for accurate quantification
real-time PCR (rtPCR or RT-PCR)
quantitative PCR (qPCR)
adapted from Real-Time PCR application guide, Bio-Rad, 2006

6. Many names, much confusion
RT-PCR = real-time PCR
Quantification
qPCR = quantitative PCR
Quantitation
RT-(q)PCR = Reverse Transcription – (q)PCR
Ct = Threshold cycle
Cq = Quantitative cycle
Cp = Crossing point
Housekeeping genes
TOP = Take-off point
Reference genes
Bustin et al., Clin Chem., 2009

7. One basic concept, many different chemistries
Fluorescent dye-based
Fluorescent primer- and probe-based
TaqMan probe assay
Molecular beacon assay
Hybridisation probes assay
SYBR Green I assay
Eclipse probe assay
HRM assay
(BEBO, Eva Green, LC Green, SYTO9)
Amplifluor primer assay
Scorpions primer assay
LUX primer assay

8. The SYBR Green I assay Advantages: Disadvantages: Cheap
Simple assay setup
Melt curve analysis to assess assay specificity
Disadvantages:
Singleplex only
Dye binding is non-specific
adapted from Real-Time PCR application guide, Bio-Rad, 2006

9. The SYBR Green I assay: amplicon and primer design
The perfect amplicon:
The perfect primers:
Length = bp C
Place Gs and Cs on ends of primers G
Tm (for both) = 59-61°C
Length = bp
No 3’ complementarity (avoids primer dimers) G A T C
Avoid stretches of single bases (< 4 bp) G C
GC content = 50-60%
No secondary structure C G A
Avoid stretches of G/C (< 3 bp)
G/C
GC content = 50-60%
No secondary structure
Tools:
For primer design (length, Tm, GC content…), Primer 3 -> or
For secondary structure prediction, mfold ->
To check primer specificity, BLAST -> or BLAT/In-silico PCR at the UCSC genome browser ->
For a database of validated primers, PrimerBank -> or RTPrimerDB ->
Real-Time PCR application guide, Bio-Rad, 2006

10. The SYBR Green I assay: what is a melt curve?
- 1st Derivative

11. The SYBR Green I assay: good vs. bad melt curves
Primer dimers!!!
Primer dimers!!!
Good melt curve

12. The SYBR Green I assay: serial dilutions of a template DNA are used for optimisation
Linearity:
Efficiency:
Pearson correlation coefficient (r)
E = 10-1/slope
Coefficient of determination (R2)
%E = (E-1) x 100%
adapted from Real-Time PCR application guide, Bio-Rad, 2006

13. QuantiTect Reverse Transcription
Kit contents:
gDNA WipeOut buffer, 7x
Quantiscript reverse transcriptase
Quantiscript RT buffer, 5x
RT primer mix
RNase-free water

14. TaqMan protocol

15. Expression quantification
Livak method:
ΔCq(sample) = Cq(target) – Cq(references)
2-Cq target
2-Cq references
Normalised expression(sample) =
Normalised expression ratio (fold change)
Normalised expression(test sample)
Normalised expression(control sample) =
Normalised expression(sample) = 2-ΔCq sample
ΔΔCq = ΔCq(test sample) – ΔCq(control sample)
Normalised expression ratio (fold change) = 2-ΔΔCq
Livak and Schmittgen, Methods, 2001
Pfaffl method:
E(target) ΔCq target (control-test)
Normalised expression ratio
(fold change) =
E(reference) ΔCq reference (control-test)
Pfaffl, Nucleic Acids Res., 2001

16. Expression quantification: a note about references

17. Reference genes
GAPDH
β-actin
β2-microglobulin
rRNA

18. MIQE
Minimum Information for Publication of Quantitative Real-Time PCR Experiments - PMID: 
Inadequate sample storage and preparation – variable results
Poor choice of primers and probes – inefficient assay performance
Inappropriate data and statistical analyses – misleading results
qPCR – quantitative real-time PCR; RT-qPCR – reverse transcription-qPCR
Quantitation cycle (Cq) not threshold cycle (Ct), crossing point (Cp) or take-off point (TOP)
Bustin et al., 2009

19. Normalisation
‘Is an essential component of a reliable qPCR assay because this process controls for variations in extraction yield, reverse-transcription yield, and efficiency of amplification, thus enabling comparisons of mRNA concentrations across different samples.’
Bustin et al., 2009

20. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

21. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

22. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

23. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

24. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

25. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines

26. The Minimum Information for publication of Quantitative PCR Experiments (MIQE) guidelines