Quantitative Polymerase Chain Reaction

Quantitative PCR or Real Time PCR (qPCR) allows for the real time analysis of targeted products in a selected sample. qPCR can be used to test for relative expression levels of gene targets, gene copy number, or SNP detection and quantification.

The TaqMan platform uses a combination of probes, which bind to multiple points on exposed DNA/cDNA and specifically designed target primers. The probes contain two elements bound to the 5’ and 3’ end, at the 5’ end is the reporter and the 3’ end the quencher, when these two molecules are in close contact the quencher prevents the excitation of the reporter by absorbing light energy through fluorescence resonance energy transfer. The reporter can only produce signal when the probe is broken, separating the reporter and quencher. During a reaction, polymerase will begin elongation from a primer strand and upon contact with a probe will cleave the molecule separating the reporter from the quencher. The reporter can then fluoresce, free of the quencher, providing a permanent increase in fluorescence, which represents the doubling of the target sample. For gene expression profiling, a method termed relative quantification is used, gene expression in the selected sample is compared against the other samples in the assay. The comparison is performed by comparing the comparative threshold cycles (Ct) values. Ct denotes the PCR cycle number at which the fluorescence signal crosses the threshold barrier, or the point where significant and specific target amplification takes place.

The SYBR Green assay measures targeted product increase by binding of the reporter dye exclusively to double stranded DNA. The dye only fluoresces when

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bound to double stranded DNA, and intensity increases with each cycle as more of the dye is bound. This requires high specificity when designing experiments as unspecific primer binding and primer dimers can create false positive results. The use of melting curves allows for the detection of multiple amplicons, suggesting unspecific binding in the assay. For copy number analysis with the SYBR green platform the absolute quantification method is used, where a standard curve is generated using known DNA dilutions, this can then be used to analyse unknown samples.

2.3.9.1 SYBR Green qPCR Primer Design

Gene sequences were identified using http://www.ncbi.nlm.nih.gov/ genome data base and primers were designed to cover potential transcript variant and exon- exon boundaries. Sequences were pasted into PrimerPlus3 to produce amplicons of 50-150bp, contain a G/C content between 30-80% and a melting temperature of 58-60o_{C. The resulting primer sequences were then validated by} BLAST and BLAT to determine specificity. Lysophilised primers were ordered from Sigma at a concentration of 100µmol, resuspended with dH2O and stored at -80o_{C, working concentration at 10µmol was stored at -20}o_{C. Primer dimer} formation was tested by investigating resulting melt curves, which indicate the presence of none specific product amplification. Upon the detection of multiple peaks, when investigating melt curves, different primer combinations were used when multiple primer sets for identical targets were available or different primer concentrations were tested. If both of these steps failed to produce one clean single peak of amplified material, new primers were designed.

2.3.9.2 SYBR Green qPCR Platform

SYBR Green master mix was created using reagents in Table 2.7 for each target gene, SYBR green contains ROX reference dye which reduces background noise by normalising non-PCR fluorescence variation. Samples were analysed in 384 well plates with 6ul of master mix pipetted per well and 4µl of 1ng patient DNA (4ng) added for a total of 10µl per well. Each well was repeated in triplicate. A standard curve is used to quantify unknown sample expression against a known set of standards, standard curves were generated for each primer target. A

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standard master mix solution was used with dilutions of control DNA purchased from Promega. DNA dilutions used to create standard curves varied but the most common dilution combination was 100ng, 75ng, 50ng, 10ng, 7.5ng and 5ng. H2O negative controls were created for each primer combination. When completed an adhesive cover was placed over the plate, which was then briefly centrifuged to remove air bubbles in individual wells. The plate was then placed into the ViiA 7 Real-Time PCR machine and analysed using the standard curve program settings. Reagents Volume SYBR Green qPCR Master Mix 5.1µl Forward Primer 10µM 0.2µl Reverse Primer 10µM 0.2µl H2O 0.5µl

Table 2.7. Composition of SYBR Green master mix per well.

2.3.9.3 TaqMan Platform

TaqMan qPCR setup was highly similar to SYBR Green qPCR. Master Mix components differed (Table 2.8) and 5ng of cDNA was used for the sample template for a total of 20µl of solution per well. Experimental plates were created with triplicate wells per reaction and negative RNAse Free H2O control wells per gene target. When placing into the ViiA 7 Real-Time PCR machine plates were analysed using the Delta CT program settings.

Reagents Volume

Gene Expression qPCR Master Mix

10µl

Probe / Primer Mix 1µl

cDNA (1ng/µl) 5µl

RNAse Free H2O 4µl

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