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Performance Comparisons of Commercial RT-PCR Systems

targets. However, product yields were consistently higher with the ProSTAR ultra HF RT-PCR system under more demanding conditions, where longer, low-abundance targets are amplified (Figure 2, Panel C). Using Advantage RT-for-PCR kit reagents, successful amplification was not achieved for any of the targets we tested.

prostar Ultra HF RT-PCR System Provides the Most Accurate RNA Amplification

The accuracy of the ProSTAR ultra HF RT-PCR system was compared to other commercial RNA amplification kits. A PCR-based forward mutation assay4 was modified to allow amplification of the lacI target gene from cDNA synthesized from in vitro-transcribed mRNA. The lacI-containing RT-PCR products were produced with various RNA amplification systems/kits using conditions that were as similar as possible, while following the guidelines specified by each manufacturer (Figure 3 legend). RT-PCR products were cloned into lambda arms, and the percentage of lacI mutants was determined in a color-screening assay. Separate controls, in which the RT was omitted, were performed for each kit. Results indicated that the RT-PCR products used in the fidelity assay were synthesized from reverse-transcribed cDNA, not from contaminating genomic DNA template.

Fig.3

Of the RT-PCR systems/kits tested, mutation frequencies were found to be lowest in RT-PCR reactions carried out with the ProSTAR ultra HF RT-PCR system (Figure 3). The percentage of mutant clones was significantly greater for all other RNA amplification systems/kits, irrespective of the reverse transcriptase, cDNA synthesis temperature, or kit format employed. The
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