Reverse transcription PCR (RT-PCR) is a technique used to detect and amplify RNA. It combines two steps: reverse transcription of RNA into complementary DNA (cDNA), followed by PCR amplification of the cDNA. This allows scientists to study gene expression and detect RNA viruses.
How RT-PCR Works
- RNA Extraction
Total RNA is extracted from the sample and treated with DNase to remove any contaminating genomic DNA. The RNA quality and concentration are checked before proceeding.
- Reverse Transcription
The RNA is mixed with reverse transcriptase enzyme, random primers or oligo-dT primers, and nucleotides. The reverse transcriptase uses the RNA as a template to synthesize a complementary DNA strand. The RNA template is then degraded by RNase H, leaving single-stranded cDNA.
- PCR Amplification
The cDNA is then used as the template for standard PCR with gene-specific primers. The PCR amplifies the cDNA, producing enough DNA to visualize on a gel or quantify by qPCR.
- Detection
The amplified PCR products are analyzed by agarose gel electrophoresis. The presence of a band at the expected size confirms that the target RNA was present in the original sample.
- Applications
RT-PCR is widely used to measure gene expression levels, detect RNA viruses such as HIV and SARS-CoV-2, validate RNA sequencing data, and analyze alternative splicing. When combined with qPCR, it becomes RT-qPCR, allowing real-time quantification of RNA.
Practical RT-PCR Protocol
Begin by extracting total RNA using a silica-column or TRIzol-based method. Assess RNA integrity by agarose gel electrophoresis or a bioanalyzer — intact RNA shows sharp 28S and 18S ribosomal bands with a 28S:18S ratio near 2:1. Measure concentration and purity by UV spectrophotometry (A260/A280 > 1.8, A260/A230 > 2.0). Treat 1–5 µg of RNA with DNase I at 37°C for 30 minutes, then heat-inactivate at 75°C for 10 minutes. For reverse transcription, mix the DNase-treated RNA with random hexamers or oligo-dT primers (50–250 ng), 1 mM dNTPs, and 200 U of reverse transcriptase in the supplied buffer. Incubate at 25°C for 10 minutes, 42°C for 50 minutes, and 70°C for 15 minutes. Dilute the resulting cDNA 5–10 fold before PCR amplification. Always include a no-reverse-transcriptase control (no-RT) to detect genomic DNA contamination — if the no-RT sample produces a PCR product, residual DNA is present. For gene-specific primers, design them to span an exon-exon junction to avoid amplifying genomic DNA; a forward primer on one exon and a reverse primer on a downstream exon ensures that only spliced cDNA is amplified.
Real-World Application
In SARS-CoV-2 diagnostics, RT-PCR targets the viral N, E, and RdRp genes using CDC or WHO primer-probe sets. RNA is extracted from nasopharyngeal swabs, and RT-qPCR with dual-labeled probes detects viral RNA at cycle thresholds (Ct) below 40. A positive result in two of three gene targets confirms infection. The inclusion of an internal control (e.g., human RNase P) verifies sample adequacy and extraction success, preventing false negatives from poor sample collection.
