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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

CTB, were always run in parallel for each cDNA template in each experimental run as a reference for accuracy of sample dilution (even if not shown in figure).


Results and Discussion
Experiment 1: Performance Over Time of Mix A Protocol Optimized for Real-Time RT-PCR
Reactions were carried out using the GSPs that amplified a 128 bp fragment of the macula-enriched transcript of interest. The amplification curve for the macula-derived sample crossed a threshold of 100 relative fluorescence units (RFU) after 21.5 cycles, and the periphery-derived sample crossed this threshold 1.7 cycles later at 23.2 cycles. These results confirmed that the transcript of interest was, indeed, enriched in the macula compared to the rest of the retina (Figure 1A). When the experiment was repeated one month later using the same reaction components, only the ACTB-derived PCR products were generated; none of the 128 bp target was detected (data not shown). The same set of real-time RT-PCR reactions was prepared again with previously unopened aliquots of each reagent stored at 20C in a constant-temperature freezer. Again, only the ACTB-derived transcripts were amplified (Figure 1B). Traces for late amplifications (CT > 34) of the 128 bp primer set represent primer-dimers and not specific product, as determined by melt-curve and gel analysis (not shown). These results showed that failure to amplify was not due to freeze-thaw induced deterioration of the stock reagents over time and suggested that some component of the stock reagents was unstable over time, even at 20C.

Experiment 2: Comparison of Reactions Based on iQ Supermix vs. Mix A
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