One-Step/Two-Step RT-PCR

Reverse transcription and PCR can be conducted in a single buffer, PCR following reverse transcription (One-Step RT-PCR) or in two separate reactions (Two-Step RT-PCR). The advantages of One-Step RT-PCR are speed and reduced contamination risk; the disadvantages are wasteful use of precious RNA and inability to control cDNA biases independently  (Bustin, 2000; Bustin et al., 2009). These (dis)advantages are reversed for Two-Step RT-PCR, with the additional advantage that the cDNA can be used for many other purposes as well. Two-Step RT-PCR also tends to be considerably more sensitive and more prone to artefacts, unless steps are taken to avoid this (see the BEEBOOK paper on molecular methods; Evans et al., 2013).