1. Grow 500 ml of yeast in YPD medium to OD600 = 0.4-0.8.
The protocol can be scaled down to as little as 10 ml of yeast.
2. Pellet cells by centrifugation. Decant supernatant, and resuspend
cells in 20 ml of the lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.5% SDS).
3. Cell pellets can be frozen in liquid nitrogen immediately
and stored at -80 °C.
4. Add equal volume of acid phenol (water-saturated), vortex.
5. Incubate at 65 °C for 60 minutes with shaking.
6. Chill on ice. Separate phases by centrifugation.
7. Transfer the aqueous phase to a fresh tube.
8. Extract again with equal volume of acid phenol (water-saturated), vortex.
9. Separate phases by centrifugation.
10. Transfer the aqueous phase to a fresh tube. Add 1/10 volume of sodium acetate (pH 5.2),
and 2.5 volumes of ethanol.
11. Precipitate at -20 °C for > 30 minutes.
12. Pellet RNA by centrifugation.
13. Wash the pellet with 70% ethanol, air dry, and resuspend the RNA in
2-5 ml of RNase-free TE (pH 7.4).