4.7.1. One-Step versus Two-Step RT-PCR
The buffer conditions for reverse transcription and PCR are largely compatible, which means that the two steps can be coupled in a single tube reaction, with the incubation conditions favouring first the reverse transcription, and then the PCR. Such ‘One-Step’ RT-PCR kits reduce the number of manipulations and associated errors, both qualitative and quantitative. The disadvantage is that they use up the sample RNA at a much higher rate than ‘Two-Step’ RT-PCR, where the cDNA is produced independently in a separate reaction. One-Step RT-PCR is also generally less sensitive than Two-Step RT-PCR, since the reaction conditions are not optimised exclusively for reverse transcription, and cannot account easily for variable reverse transcription efficiencies between different assays/primers (Bustin, 2000; Bustin et al., 2009). The main disadvantage of Two-Step RT-PCR is that the additives included in the reverse transcription buffer to enhance primer binding and reaction efficiency, can also encourage the production of non-specific PCR products during PCR, which affects the quantitation accuracy. To minimize such effects, cDNA should be diluted ten-fold with water before being used for Two-Step RT-PCR.
Commercial One-Step or Two-Step RT-qPCR kits have proprietary reagent mixtures that are optimised for the corresponding recommended cycling profiles. Different kits therefore perform differently with particular primers and cycling profiles (Grabensteiner et al., 2001), and the choice of RT-PCR kit is therefore also part of the optimization procedure. To take maximum advantage of such pre-optimized systems, the most practical approach is to design the assays and primers to fit these optimized recommendations, whenever this is possible.