9.3.3.1. Reverse transcription

Reverse transcription is the most variable step in RT-PCR, whose efficiency is easily affected by inhibitors, reaction conditions (including primers) and even nucleic acid concentration (Ståhlberg et al., 2004a; 2004b). To minimize this variability, the nucleic acid should be optimally prepared and a constant amount used in every reaction. Since PCR does not require large amounts of initial target, the RNA can be diluted to minimise the effects of any inhibitors. cDNA is best prepared with random hexamer primers that generate a bias-free cDNA copy of the entire RNA population, suitable for a multitude of analyses.