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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

dsized PCR products were then 23.4 1.9 for the macula and 24.4 1.3 for the periphery, for an average 1.0 cycle difference (Figure 4B). Clearly, the real-time RT-PCR results generated using the iQ supermix were more reliable and reproducible.


Conclusions
Although quantitative real-time RT-PCR is a powerful, sensitive, and reproducible method to quantitate differences in mRNA expression, many aspects of the reactions (i.e., primer design, annealing temperatures, and master mix reagents) must be optimized to put this powerful technology to work successfully in a new experimental system. Realtime RT-PCR is especially sensitive to product length, where longer length products and low- to medium-abundance transcripts cannot be amplified in reactions containing unstable reagents. While Bio-Rads bulletin 2593 (Boeckman et al. 2001) for the iCycler thermal cycler recommends amplification of PCR products only within the narrow range of 75150 bp, we have consistently been able to amplify products in excess of 300 bp using iQ supermix (data not shown). Replicate CT values for amplification of the control gene ACTB showed less variation between replicates and between experiments with either reaction mix (Figures 1 and 3). This was not simply due to the relative abundance of ACTB transcripts in the samples since the amount of the macula-enriched target gene was the same as ACTB in the macula. Instead, these results suggest that sequence-related secondary structure or transcript stability of the target gene could affect outcomes in the mix A reactions but were not a factor in the iQ supermix reactions. Finally, the iQ supermix reactions were not only more robust but also w
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