Reverse transcription polymerase chain reaction

Reverse transcription polymerase chain reaction (RT-PCR), a variant of polymerase chain reaction (PCR), is a technique commonly used in molecular biology to detect RNA expression. RT-PCR is often confused with real-time polymerase chain reaction (qPCR) by students and scientists alike, but they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent dyes. qPCR is also referred to as quantitative PCR, quantitative real-time PCR, and real-time quantitative PCR.

Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

In addition to the qualitative study of gene expression, quantitative PCR can be utilized for quantification of RNA, in both relative and absolute terms, by incorporating qPCR into the technique. The combined technique, described as quantitative RT-PCR or real-time RT-PCR (sometimes even quantitative real-time RT-PCR), is often abbreviated as qRT-PCR, RT-qPCR, or RRT-PCR. Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.

In order to avoid confusion, the following abbreviations will be used consistently throughout this article:

Since its introduction in 1977, Northern blot had been used extensively for RNA quantification despite its shortcomings: (a) time-consuming technique, (b) requires a large quantity of RNA for detection, and (c) quantitatively inaccurate in the low abundance of RNA content. However, the discovery of reverse transcriptase during the study of viral replication of genetic material led to the development of RT-PCR, which has since displaced Northern blot as the method of choice for RNA detection and quantification.

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