Reverse transcription-polymerase chain reaction (RT-PCR) converts and amplifies a single-stranded RNA template to yield abundant double-stranded DNA product(1,2,3). RT-PCR is a powerful tool for analysis of gene expression and for characterization of RNA splice variants. RT-PCR is generally carried out as either one-step RT-PCR, in which all reagents are combined in one tube and RT and PCR are conducted sequentially in this tube(1,3) or two-step RT-PCR, in which RT and PCR are conducted sequentially in separate tubes(2). The USB One-Step RT-PCR Kit (PN 78350), Two-Step RT-PCR Kit (PN 78355), and RT Script Kit (PN 78360) are based on M-MLV Reverse Transcriptase(4) and Taq DNA Polymerase(5). M-MLV Reverse Transcriptase is useful in RT-PCR due to its naturally low RNase H activity(2). The kits are designed for typical routine analyses of gene expression, based on amplification of relatively short (≤ 1.5 kb) RT-PCR products (Fig. 1).
Optimization of reaction parameters is a crucial aspect of RT-PCR. With new targets, optimization can determine the difference between failure and success in generating product. With familiar targets, optimization can minimize use of sample and reagents and maximize reproducibility and reliability of results. A small investment of effort in optimizing reactions can yield a large payoff in results, particularly if careful attention is also paid to fundamental issues such as primer design(6,7), RNA isolation(8,9), and use of RNase free reagents(10). This Tech Tip describes several simple approaches for optimizing sensitivity, specificity, and yield in one-step RT-PCR. The same approaches also apply in two-step RT-PCR.
Reactions (50 μl) were carried out with the USB One-Step RT-PCR Kit (PN 78350) according to the kit protocol, except as no