RT-PCR

Reverse Transcription Polymerase Chain Reaction

Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase chain reaction (PCR). More importantly it is based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. 

The techniques of RT-PCR allows the formation of cDNA (complementary or copy DNA) from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the more stable form of nucleic acid, DNA. This reverse transcription from RNA into its reverse complement DNA (cDNA) is the first step of a usually two-step process of RT-PCR. Furthermore, by copying the RNA into DNA, one can then amplify the cDNA sequence by using primers specific for the DNA sequence. This amplification is the final second major step of the two-step process of RT-PCR.

The Process of RT-PCR

The First step of RT-PCR is referred to as the "first strand reaction". In the first-strand reaction, complementary DNA also termed cDNA, is made from the messenger RNA template of interest using oligo dT (oligonucleotide poly-dTs act similar to primers and bind to the 3' polyA sequence located at the 3' UTR - untranslated region, which are present in most mRNAs), dNTPs, and an RNA-dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription.

These factors are combined in a reverse transcriptase buffer for 1 hour at 37°C. After reverse transcriptase reaction is complete, and the cDNA has been synthesized, RNaseH is added (an RNA digestion enzyme) which digests the RNA away from the RNA-cDNA hybrid. After incubation with RNaseH, standard PCR or polymerase chain reaction is conducted using DNA oligo primers specific for the sequence of interest. This second step is referred to as the "second strand reaction".

Thus by adding the thermostable DNA polymerase, upstream and downstream DNA primers, the single stranded DNA becomes double stranded and is amplified, allowing the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

Applications of RT-PCR

The exponential amplification of complementary sequence of mRNA or RNA sequences via reverse transcription polymerase chain reaction allow for a high sensitivity detection technique, where low copy number or less abundant RNA molecules can be detected. It is also used to clone mRNA sequences in the form of complementary DNA, allowing libraries of cDNA (cDNA libraries) to be created which contain all the mRNA sequences of genes expressed in a cell. Furthermore, it allows the creation of cDNA constructs which were cloned by RT-PCR and allow the expression of genes at the RNA and protein levels for further study.