Reverse transcription PCR (RT-PCR)

Principle

• RNA cannot be amplified directly by Taq polymerase, so it is first copied into complementary DNA (cDNA) by reverse transcriptase (e.g. from MMLV or AMV retroviruses).
• cDNA is then amplified by PCR, often in real time (RT-qPCR). Note: "RT" here means reverse transcription, not real-time.
• RT priming: oligo(dT) (binds poly-A tails of mRNA), random hexamers (all RNA) or gene-specific primers.

Specimen

• Nasopharyngeal or oropharyngeal swabs in transport medium, EDTA plasma (HIV, HCV viral load), bone marrow or blood (fusion transcripts), tissue.
• Process quickly, keep cold, and freeze at –70 °C or below for long storage.

Procedure

• Extract RNA with RNase-free technique.
• One-step RT-PCR: RT and PCR in one tube (less handling, lower contamination risk). Two-step: separate RT reaction, cDNA reusable for several targets.
• Typical RT incubation about 42–50 °C for 10–30 minutes, then 95 °C to inactivate RT and activate the polymerase, followed by PCR cycling.

Quality control and pitfalls

• No-RT control detects genomic DNA contamination; DNase treatment and primers spanning exon–exon junctions reduce it.
• Include negative, positive and internal controls (e.g. human RNase P gene to confirm adequate sampling, or an added exogenous control).
• RNA degradation gives false negatives; poor swab technique is a common cause.
• Detecting RNA does not prove live virus; RNA may persist after recovery.

Clinical use

• RNA viruses: SARS-CoV-2, influenza, HIV, HCV, dengue, measles.
• Fusion transcripts: BCR-ABL1 in CML and ALL, PML-RARA in acute promyelocytic leukaemia.
• Gene expression studies.