Before you start
- Clean the bench and pipettes with an RNase decontaminant; wear gloves throughout.
- Pre-cool the centrifuge to 4 °C.
Reagents and materials
Totals are for 12 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| TRIzol (per 5–10 × 10⁶ cells or 50–100 mg tissue) | 1 mL | 1 mL |
| Chloroform | 0.2 mL | 0.2 mL |
| Isopropanol | 0.5 mL | 0.5 mL |
| 75% ethanol in nuclease-free water | 1 mL | 1 mL |
| Glycogen (RNase-free, optional carrier)for small samples | 1 µL | 1 µL |
| Nuclease-free water | 30 µL | 30 µL |
Equipment
- Fume hood
- Refrigerated microcentrifuge (12,000 × g)
- RNase-free tubes and filter tips
- Homogeniser for tissue
- Spectrophotometer (NanoDrop) and optional Bioanalyzer/TapeStation
Procedure
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Homogenise
Lyse cells (or homogenise tissue) in 1 mL TRIzol and incubate 5 min at room temperature. Lysates can be stored at −80 °C at this point.
Add chloroform
Add 0.2 mL chloroform, shake vigorously for 15 s and incubate 2–3 min at room temperature.
Separate phases
Centrifuge at 12,000 × g for 15 min at 4 °C.
Transfer aqueous phase
Move the clear upper phase to a new tube without touching the interphase.
Precipitate
Add 0.5 mL isopropanol (plus glycogen for small samples), mix and incubate 10 min; centrifuge at 12,000 × g for 10 min at 4 °C.
Wash
Remove supernatant, add 1 mL 75% ethanol, vortex briefly and centrifuge at 7,500 × g for 5 min at 4 °C.
Dry
Remove ethanol and air-dry 5–10 min. Do not over-dry.
Dissolve
Dissolve in 20–50 µL nuclease-free water (optionally 10 min at 55–60 °C).
Check quality
Measure concentration; aim for A260/280 ≈ 2.0 and A260/230 > 1.8. Check integrity (RIN) for sequencing. Store at −80 °C.
Critical points
- DNase-treat (on-column or in solution) before qPCR with primers that do not span introns.
- Low A260/230 means guanidine or phenol carry-over: re-precipitate or clean up on a column.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low yield | Use enough starting material, homogenise fully, add glycogen carrier. |
| Degraded RNA | Work faster and colder, use RNase-free consumables, store lysate at −80 °C. |
| Low 260/230 | Avoid the interphase; extra ethanol wash; column clean-up. |
Safety
TRIzol contains phenol and guanidine thiocyanate; chloroform is toxic. Work in a fume hood with nitrile gloves; collect organic waste. Never mix TRIzol waste with bleach.
Further reading
- Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem (1987).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.