Good qPCR The Necessary and the Reasonable

1993 Nobel Prize in Chemistry PCR origins 1993 Nobel Prize in Chemistry

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

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

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

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

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

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

The SYBR Green I assay: amplicon and primer design The perfect amplicon: The perfect primers: Length = 19-21 bp C Place Gs and Cs on ends of primers G Tm (for both) = 59-61°C Length = 75-200 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 -> http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi or http://primer3.ut.ee For secondary structure prediction, mfold -> http://www.bioinfo.rpi.edu/applications/mfold/ To check primer specificity, BLAST -> http://www.ncbi.nlm.nih.gov/blast/ or BLAT/In-silico PCR at the UCSC genome browser -> http://genome-euro.ucsc.edu For a database of validated primers, PrimerBank -> https://pga.mgh.harvard.edu/primerbank/ or RTPrimerDB -> http://www.rtprimerdb.org Real-Time PCR application guide, Bio-Rad, 2006

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

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

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

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

TaqMan protocol

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

Expression quantification: a note about references

Reference genes GAPDH β-actin β2-microglobulin rRNA

MIQE Minimum Information for Publication of Quantitative Real-Time PCR Experiments - PMID: 19246619 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

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

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

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

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

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

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

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

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