In the direct one-step method, there is very little sample processing. The extraction process generally is done in the PCR tube. Eg. reactions from blood samples can be run directly. The reverse transcriptase is also included in the same tube as the PCR. This saves time and reduces the possibility of sample contamination. A two step reaction will have better sensitivity.

The one-step method requires RNA isolation prior to running the PCR reaction. The reverse transcriptase and PCR reactions are run in the same tube. This saves time and reduces the possibility of sample contamination. A two step reaction will have better sensitivity.

The two-step method requires RNA isolation, next a reverse transcriptase reaction and then the actual PCR reaction can be done. Two-step RT-PCR is highly sensitive, potentially more efficient, and allows for stocking of the cDNA for future use. However, this method is more time consuming and can require more optimization.