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Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A

f iQ supermix rescued the assay, resulting in accumulation of the macula-derived 128 bp products crossing the threshold of 100 RFU after 19.7 cycles (Figure 2) almost 2 cycles earlier than in the mix A-based reactions with macula cDNAs for template (Figure 1). In the iQ supermix reactions containing periphery-derived cDNAs, the 128 bp product crossed this threshold value at 21.2 cycles (Figure 2), 1.5 cycles later than the macula reactions with the iQ supermix and almost 2 cycles earlier than with the mix A periphery reactions in experiment 1 (Figure 1). Equivalent mix A reactions run at the same time failed to amplify (Figure 2). As shown in the following experiment, the reproducibility of these results as well as the stability of the iQ supermix reagents held up over time.

Experiment 3: Performance of iQ Supermix Reactions Over Time
Real-time RT-PCR was performed as described for experiment 2, except that the iQ supermix (2x) stock used in these reactions had been stored for 4 months at 20C. The 128 bp segment of the macula-enriched transcript was again successfully amplified. The amplification curve for the macula sample crossed the threshold of 100 RFU after 20.1 cycles, while the periphery sample crossed the threshold 1.7 cycles later at 21.8 (Figure 3). The iQ supermix is therefore more stable over time than mix A.

Experiment 4: Amplification of a Specific Transcript Using Three Different Pairs of GSPs With iQ Supermix vs. Mix A
In order to test whether primer design can affect the reproducibility of amplification curves obtained for a specific transcript in a specific tissue, real-time RT-PCR was performed using three pairs of primers designed to amplify di
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