Do the Two-Step

Bitesize Bio covers the advantages that two-step RT-PCR reactions have over one-step qRT-PCR. These include reduced formation of primer dimers, reduced variability between two different RT reactions, less need to perform multiple reactions for housekeeping genes, increased flexibility in the amount of reverse transcriptase added to the reaction, and little intra-assay variation. In any case, the author recommends taking breaks for quality control purposes. "They allow you to take stock and examine your results to see if things are still on track, and makes troubleshooting far easier. Even just for this reason, I am sold on two-step, rather than one-step, qRT-PCR."