RNA extraction phenol–guanidine method
Cells are lysed in a phenol–guanidine reagent; adding chloroform splits the mixture into layers and RNA stays in the clear upper aqueous phase. It is then precipitated with isopropanol. Preventing RNase and not disturbing the layers is everything.
Key points
- RNase-free tools and gloves; lyse samples quickly
- Take only the upper aqueous phase — touching the interphase adds DNA and protein
- Check purity with A260/280 (≈2.0) and A260/230 (2.0–2.2)
Materials
- Phenol–guanidine reagent (TRIzol-type)
- Chloroform
- Isopropanol
- 75% ethanol (made with RNase-free water)
- RNase-free water
- RNase-free tips and tubes
- Refrigerated centrifuge (4 °C)
- Spectrophotometer (NanoDrop-type)
Steps
- 💡 You can store the lysate at −80 °C here and continue later.
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Starting material
Aqueous phase recovery
Purity (A260/280 · A260/230)
Pellet drying
Method comparison
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Low yield | Too little sample, incomplete lysis, lost pellet | Homogenise thoroughly, add a carrier, decant carefully |
| Low A260/280 (<1.8) | Phenol or protein contamination | Take less aqueous phase, re-extract with chloroform |
| Low A260/230 (<1.5) | Residual guanidine or phenol | Extra 75% ethanol wash, re-precipitate |
| Degraded RNA (smear on gel) | RNase contamination, samples left sitting | Lyse immediately, work on ice, RNase-free tools |
| Genomic DNA contamination | Interphase carry-over | DNase treatment, intron-spanning primers |
⚠ Safety Phenol and chloroform are toxic and burn skin — use a fume hood, nitrile gloves and eye protection, and collect waste separately.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
A260 concentration & purity → qPCR analysis (ΔΔCt) →