24: Reverse transcription PCR
Summary
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 r eal t ime 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