What is RT efficiency?

RT efficiency refers to how accurately the RT enzyme can represent the number of RNA molecules as cDNA in reverse transcription (RT). Understanding RT efficiency and how to calculate it is vital for interpreting data accurately.

The ratio of the number of cDNA templates that are generated from a given number of RNA transcripts for a given gene is expressed as a percentage and is referred to as RT efficiency. For example, if a particular sample that contains 1,500 RNA transcripts for a given gene generates exactly 1,500 cDNA templates, the reaction is said to have a 100% RT efficiency.

Contrary to common assumption, all the RNA/mRNA in a RT reaction is not converted to cDNA and all transcripts are not converted in a 1:1 ratio or proportionally to the starting RNA concentration. In practical applications, the amount of RNA that gets converted into cDNA is highly variable and in most cases RT efficiency is below 100%.  Bad primer design and non-optimal reagent concentrations or reaction conditions are the most common reasons for lower RT efficiencies.