What are you going to learn?

  • What is RT-PCR?
  • How does RT-PCR work?
  • What are the differences between standard PCR and RT-PCR?
  • What are the applications of RT-PCR?

RT-PCR (reverse transcriptase polymerase chain reaction) is another modification of standard PCR, which uses an enzyme called reverse transcriptase to transcribe RNA into cDNA, which is then amplified using the standard PCR method. Reverse transcriptase is mainly found in retroviruses (e.g., HIV), which, after infecting a host cell, use the enzyme to transcribe their RNA into cDNA, which is then incorporated into the genome of the infected cell.

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RT-PCR is a PCR modification, which uses reverse transcriptase to transcribe RNA into cDNA.
reverse transcriptase and its function

Apart from reverse transcriptase and RNA template, RT-PCR uses the same basic components as conventional PCR: DNA polymerase, primers, dNTPs and a buffer.

components needed for RT-PCR

The carry out an RT-PCR, first, a primer must bind to the RNA molecule, which allows reverse transcriptase to synthesize a new cDNA strand. The original RNA is then degraded by another enzyme RNAse H and a second cDNA chain is synthesized by DNA polymerase.

reverse transcription in RT-PCR

Then the standard PCR steps follow: the individual chains are separated (denaturation); the primers bind to specific complementary sequences (annealing) and the DNA polymerase synthesizes the new chains (extension). This process is repeated many times, resulting in a huge increase in the reaction product.

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RT-PCR steps:
1) primer binds to RNA
2) reverse transcriptase synthesizes a cDNAasfd
3) the original RNA is degraded
4) DNA polymerase synthesizes a second cDNA strand
5) standard PCR - denaturation, annealing, extension
standard PCR in detail

Applications

Today, perhaps the most well-known application of RT-PCR is the diagnosis of diseases caused by RNA viruses. The viral RNA is transcribed into cDNA by reverse transcriptase and amplified after complementary primer bind to their target sequence. The amplified viral cDNA then confirms the viral infection. However, there are many other applications of RT-PCR. For example, RT-PCR is used to analyse gene expression in cells. The mRNA molecules are converted into cDNA, and these can then be analyses.

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RT-PCR is often used in viral diagnostics.

References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.
Tubbs, R. R., & Stoler, M. H. (2009). Cell and Tissue Based Molecular Pathology. Churchill Livingstone/Elsevier.