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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

fferent regions of the same target transcript. The three GSP pairs generate 128 bp, 100 bp, and 99 bp products. Duplicate reactions using each primer pair with each reaction mix (A or iQ supermix) were run for each template. The performance of the iQ supermix reactions was quite consistent for each primer pair (Figure 4A), whereas the performance of the mix A-based reactions varied for each GSP (Figure 4B).

In the iQ supermix reactions, the average CT for all six traces representing the amplification of the macula sample with three different primer pairs was 19.9 0.3 cycles. The average CT for the periphery-derived sample was 21.4 0.2 cycles, resulting in an average of a 1.5 cycle difference between the two regions of human neural retina (Figure 4A). This differential expression profile for the macula-enriched gene transcript was the same as that obtained in the two previous experiments with iQ supermix reactions (Figures 2 and 3).

The traces representing the accumulation of PCR products in the mix A-based reactions varied with each GSP (Figure 4B), in contrast to the traces for the iQ supermix reactions (Figure 4A). In the mix A-based reactions, the 128 bp fragment was not amplified at all, whereas the products generated by the other two primer pairs were amplified at different rates. The amplification curves for the 100 bp fragment crossed threshold fluorescence at 25.4 cycles in the macula reactions and 25.8 in the periphery, while the curves for the 99 bp fragment from closer to the 3' end of the target mRNA crossed the threshold at 21.5 cycles in the macula reactions and 23.1 in the periphery. The average CT values for the two GSP pairs that resulted in the expecte
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