RT-PCR

RT-PCR or Reverse transcription polymerase chain reaction is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

Procedure

The procedure for RT-PCR consists of two parts: the synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and the amplification of a specific cDNA by the polymerase chain reaction (PCR).

Reverse Transcription

In the first step of RT-PCR, the enzyme reverse transcriptase synthesizes a molecule of cDNA based on an RNA template. The reverse transcriptase enzyme synthesizes the cDNA by extending the primer, which binds to the RNA template at a complementary site.

Polymerase Chain Reaction

After the cDNA has been synthesized, the RNA is degraded and the cDNA is used as a template for the polymerase chain reaction. The PCR amplifies the cDNA, creating enough of it for analysis and detection.

Applications

RT-PCR is widely used in the diagnosis of genetic diseases and in advanced biomedical, genetic, and forensic research. It is also used in the diagnosis of viral infections, including COVID-19.

See Also

• Polymerase chain reaction
• COVID-19 testing
• Molecular biology

RT-PCR Resources
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