Reverse Transcriptase PCR (RT-PCR) is a variation of the polymerase chain reaction that amplifies target RNA. Addition of reverse transcriptase (RT) enzyme prior to PCR makes it possible to amplify and detect RNA targets.
Reverse transcriptase enzyme transcribes the template RNA and forms complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. These DNA molecules can now be used as templates for a PCR reaction.
Nowadays, single thermostable DNA polymerase that also possesses significant reverse transcriptase activity is used in the single-step reaction.
Principle of RT-PCR
Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. In both cases, RNA is first reverse-transcribed into cDNA, which is then used as the template for PCR amplification.
The primers used for cDNA synthesis can be either non–sequence-specific primers (a mixture of random hexamers or oligo-dT primers) or sequence-specific primers.
- Non-sequence-specific primers:
- Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool.
- Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules.
- Sequence-specific primers:
- Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process.
cDNA synthesis and PCR are performed in a single reaction vessel in a common reaction buffer. Gene-specific primers direct cDNA synthesis and amplification of a specific target. Major advantages of one-step reaction include minimal sample handling, reduced bench time, and closed-tube reactions, reducing chances for pipetting errors and cross-contamination.
The quality and scarcity of RNA samples impact the efficiency of one-step RT-PCR. cDNA synthesis product cannot be saved after one-step RT-PCR so additional aliquots of the original RNA sample(s) are required in order to repeat reactions or to assess the expression of other genes.
In two-step RT-PCR, cDNA synthesis is carried out using random hexamers, oligo-dT primers, and/or gene-specific primers which gives a mixture of cDNA molecules. cDNAs thus synthesized are amplified using specific primers.
In two-step RT-PCR, cDNA is synthesized in one reaction, and an aliquot of the cDNA is then used for a subsequent PCR experiment. This requires extra open-tube step, more pipetting manipulations, and longer hands-on time which may lead to greater variability and risk of contamination. Remaining cDNA can be stored for future use, or quantitating the expression of multiple genes from a single RNA/cDNA sample.
Applications of RT-PCR
Many clinically important viruses have genomes composed of RNA, RT-PCR is useful for detecting such viruses. RT-PCR has also been used for the detection of the viral causes of meningitis and meningoencephalitis, such as enteroviruses and the West Nile virus. RT-PCR is being used for the detection of the following viruses:
- Dengue virus
- Human metapneumovirus
- Severe acute respiratory syndrome (SARS)
Quantitative RT-PCR assays are commonly used for the detection of HIV and HCV viral load (amount of these viruses present in the blood of a patient) testing.
Viral load data are important for monitoring the response of the individual patient to therapy. For instance, after appropriate antiretroviral therapy, patient infected with HIV virus should demonstrate an increase in CD4 count and a decrease in HIV viral load.
RT-PCR may also be used to detect other microorganisms (bacteria, parasites, and fungi) by targeting their rRNA. This approach is better than detection of DNA, as the presence of RNA is more likely associated with the presence of viable organisms.
Detection of mRNA using RT-PCR helps to study the gene expression of both microorganisms and human host cells.
References and Further Reading
- Choice of One-Step RT-qPCR or Two-Step RT-qPCR. New England BioLabs Inc.
- Molecular Cloning and Recombinant DNA Technology. Matt Carter, Jennifer Shieh, in Guide to Research Techniques in Neuroscience (Second Edition), 2015
- Molecular Anatomic Pathology. Marina N. Nikiforova, Yuri E. Nikiforov, in Diagnostic Immunohistochemistry (Third Edition), 2011