Reverse transcription (RT) is the process of converting RNA to cDNA using a reverse transcriptase enzyme and dNTPs.
The RT step may be performed on total RNA such that a global cDNA is produced that is representative of all of the RNA transcripts in the sample (usually via a two-step protocol), or in a gene-specific approach such that only the RNA of interest is converted to cDNA (usually following a one-step protocol).
The following experiments can be used as basic RT protocols that can be modified to suit particular requirements. It is customary to either prepare cDNA using a two-step process with subsequent dilution of the cDNA prior to adding it to the PCR/qPCR, or to prepare a one-step reaction where both processes are carried out sequentially.
In the example given below, the primer concentrations can be adjusted according to the results of optimization procedures (Primer Concentration Optimization, Primer Optimization Using Temperature Gradient and Assay Optimization and Validation).