1. Snap freeze ~10⁷ cells or ~100 mg of tissue in liquid nitrogen or dry ice/ethanol.

2. Transfer the frozen sample to a mortar and pestle and grind to a fine powder. Or use a hand-held tissue grinder for a microcentrifuge tube.

3. Transfer the sample dust to 1 ml of Trizol solution. Get as much the powder as possible.

4. Mix thoroughly by vortexing.

5. Further homogenize the sample by sonication or passing through an 18 gauge needle several times.

6. Add 200 ml chloroform, vortex and let sit at room temperature for 2-3 minutes.

7. Centrifuge at 12,000 rpm for 15 minutes at 4 °C.

8. Transfer the aqueous phase to a fresh tube.

9. Precipitate by adding 0.5 ml isopropanol and keep at -20 °C for at least one hour.

10. Centrifuge at 12,000 rpm for 15 minutes at 4 °C.

11. Remove supernatant carefully.

12. Wash the RNA pellet with 1 ml 80% ethanol/DEPC-ddH₂O.

13. Remove supernatant.

14. Air dry and resuspend the pellet in 50 ml of DEPC-ddH₂O.

15. Store immediately at -20 °C.