21: Reverse transcription PCR
- Page ID
- 141655
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Reverse transcription PCR is a method that is used to synthesize a complementary DNA sequence from an RNA template.
Also known as
RT-PCR
Note that the acronym RT-PCR can be confusing, as qRT-PCR (a different method) is sometimes referred to as real time PCR, so care should be taken to avoid confusion.
Samples needed
Sample of RNA, which can be a mixture of sequences
Method
The vast majority of the time, DNA serves as a template for RNA synthesis (in transcription), but not the reverse. However, the discovery of retroviruses illuminated the existence of reverse transcriptase (RT) enzymes, which use RNA as a template for DNA synthesis.
In molecular biology, RT-PCR is used to create cDNAs as an intermediate step in another experiment, or for molecular cloning. A cDNA is a “complementary” DNA, complementary to a mature mRNA. In eukaryotes, this means that a cDNA differs substantially from genomic DNA, since cDNA lacks introns.
To amplify a particular RNA sequence, two sequence-specific primers are used. However, RT-PCR is often used to create a cDNA pool of all cellular mRNAs, like in qRT-PCR. In this case, primers with random sequences can be used, or polyT primers that anneal to polyA tails of mRNAs.
Generally, the protocol for RT-PCR is very similar to that of typical PCR, with the exception that reverse transcriptase is used instead of DNA polymerase.
Controls
As with typical PCR, primer specificity should be tested. Additionally, a no template control (water instead of RNA sample) is used to ensure that none of the other reagents are contaminated with template RNA. Lastly, a no reverse transcriptase control can be used to test the presence of gDNA contaminating the RNA sample.
Thumbnail
"Reverse transcriptase 1KLF.png"↗ by Thomas Splettstoesser is licensed under CC BY-SA 3.0↗.
Description: Surface representation of the crystallographic structure of HIV reverse transcriptase based on PDB coordinates 3KLF↗.
Author
Katherine Mattaini, Tufts University