What is RT-PCR Testing?

What is RT-PCR Testing?

RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a molecular biology technique combining reverse transcription and PCR, primarily used for detecting RNA viruses or analyzing RNA expression levels. Below is a detailed breakdown of its principles, procedures, applications, and more:

I. Core Principle

The essence of RT-PCR is converting RNA into cDNA via reverse transcription, then exponentially amplifying the cDNA through PCR to enable detection or quantification of trace RNA. The core logic is: RNA → cDNA → DNA amplification → signal detection.

II. Key Steps and Technical Details

1. Sample Preparation and RNA Extraction

• Sample Types: Blood, tissues, cells, saliva, etc. (containing target RNA, such as viral RNA or mRNA).
• Extraction Methods:
  • TRIzol Method: Uses guanidine isothiocyanate to lyse cells, inactivate RNase, and extract RNA via chloroform stratification, suitable for routine samples.
  • Magnetic Bead Method: Oligonucleotide-modified magnetic beads (e.g., Oligo dT) specifically bind to mRNA, purified via magnetic separation, highly automated for large-scale testing.
• Precautions: RNA is susceptible to RNase degradation; use RNase-free consumables and store samples at low temperatures (e.g., -80°C).

2. Reverse Transcription (cDNA Synthesis)

• Reverse Transcriptase: Commonly AMV (avian myeloblastosis virus RT) or M-MLV (moloney murine leukemia virus RT), requiring dNTPs, primers (random primers, Oligo dT, or specific primers) in the buffer.
• Reaction Conditions:
  • Temperature: 37–50°C (adjusted by enzyme type, e.g., M-MLV works at lower temperatures).
  • Time: 30 min to 1 hour for RNA-to-cDNA conversion.

3. PCR Amplification

• System Components: cDNA template, Taq DNA polymerase, dNTPs, specific primers (designed for target genes), buffer (含 Mg²⁺).
• Cycle Process (30–40 cycles):
  • Denaturation: 94–95°C, 15–30 sec, to unwind DNA strands.
  • Annealing: 55–65°C, 30 sec, for primer-template binding.
  • Extension: 72°C, 30–60 sec, for Taq enzyme to synthesize new strands.
• Product Detection:
  • Gel Electrophoresis: Separate PCR products via agarose gel, stain with EB to observe target bands (for qualitative analysis).
  • Real-time Fluorescent qRT-PCR: Add fluorescent probes (e.g., TaqMan probes), which release signals during amplification for real-time quantification.

III. Main Types and Differences

IV. Application Fields

1. Medical Diagnosis

• Viral Detection: RNA viruses like SARS-CoV-2, HIV, dengue virus, hepatitis viruses.
• Genetic Disease Diagnosis: Detect mRNA abnormalities from gene mutations (e.g., cystic fibrosis, spinal muscular atrophy).

2. Molecular Biology Research

• Gene Expression Analysis: Quantify mRNA levels in cells/tissues (e.g., drug effects via RT-qPCR).
• RNA Virus Research: Analyze genome variation and replication (e.g., influenza antigenic drift).

3. Forensics and Epidemiology

• Viral Typing: Amplify conserved viral genes for sequencing to trace infection sources (e.g., COVID-19 溯源).
• Trace Sample Detection: Nested RT-PCR for RNA analysis in forensic samples (bloodstains, saliva).

V. Precautions and Limitations

1. Key Considerations

• Anti-contamination: Include negative (no template) and positive (known RNA) controls to avoid false positives from lab RNA/cDNA contamination.
• Primer Design: Target conserved RNA regions to prevent missed detection due to sequence variation (update primers for mutant viruses).
• Sample Quality: Low RNA extraction efficiency or degradation causes false negatives; assess RNA purity via spectrophotometry (A260/A280 ratio) or electrophoresis.

2. Limitations

• Window Period: Low viral RNA levels in early infection may yield false negatives (e.g., within 2 weeks of HIV infection).
• Quantification Errors: qRT-PCR results are affected by primer efficiency and reverse transcription efficiency, requiring normalization with reference genes (e.g., GAPDH).
• Operational Complexity: RNA extraction and reverse transcription require strict temperature control, demanding higher laboratory standards.

VI. Comparison with Other Techniques

• vs PCR: PCR detects DNA directly, while RT-PCR converts RNA to cDNA first, suitable for RNA viruses or expression analysis.
• vs Nucleic Acid Sequencing: RT-PCR targets known RNA sequences, whereas sequencing analyzes unknown sequences or whole genomes but is costlier and more time-consuming.