RNA Extraction
Related MOCs: MOC - Diagnostic & Lab Methods · MOC - Virology
Related: DNA Extraction · RT-PCR · Reverse Transcription Polymerase Chain Reaction (RT-PCR) · Nucleic Acid Quantification
1. Principle
RNA extraction recovers intact RNA (viral genomes, transcripts, or total RNA) while aggressively suppressing ubiquitous RNases, enabling reverse transcription and RNA-targeted NAATs.
2. Step-by-Step Procedure (conceptual)
- Inactivate RNases immediately — guanidinium chaotropes, commercial lysis buffers; RNase-free plastics/tips.
- Lyse cells/virions; for tough samples combine chemical + mechanical lysis.
- Bind RNA — silica columns or magnetic beads (many kits co-extract DNA unless DNase step added).
- Optional DNase digest — remove gDNA when assays must be RNA-specific.
- Wash → elute in RNase-free water/TE; keep cold; minimize freeze–thaw.
- Downstream: RT-PCR / qPCR (one-step or two-step), RNA-seq library prep, viral load assays.
3. Interpretation / QC
- Integrity: RIN/RQN (Bioanalyzer/TapeStation) for transcriptomics; less critical for short amplicon viral PCR.
- Pitfalls: RNase degradation → false negatives; DNA contamination → false positives in poorly designed RT-minus controls; inhibitors same as DNA preps.
4. Clinical Use Cases
- Essential for RNA viruses: influenza, SARS-CoV-2, HIV (with RT), HCV, RSV, etc.
- Bacterial pathogen transcripts / dual DNA–RNA panels.
- Research: Microbial Transcriptomics.
5. Comparison with Other Methods
| Method | When to use |
|---|---|
| DNA Extraction | DNA viruses, bacteria, WGS from isolates |
| RNA Extraction | RNA viruses, gene expression |
| Total NA kits | Syndromic panels needing both |
6. Mnemonic / Visual Aid
RNases never sleep — assume contamination until proven otherwise.
Related Concepts
Active Recall
- Why run an RT-minus control?
- When is DNase treatment mandatory?
- Why is viral RNA more fragile than DNA in swabs left at room temperature?