1. Resuspend cells in PBS to 5 x 107 cells/ml. Put on ice.
2. Add 10% SDS to a final concentration of 1%. Vortex briefly.
3. Add 1 vol. of phenol:chloroform:isoamyl alcohol (“PCI”, pH 8) and vortex for 15 sec. Incubate on ice 15 sec and then vortex again 15 sec.
4. Centrifuge tubes at 1500-2000g (~ 3000 rpm) for 5-10 min. Take aqueous phase and re-extract with 1 vol. of PCI as above.
5. Take aqueous phase and add 3 M NaAc, pH 4.7 to a final concentration of 0.4 M (154 µl per ml of aqueous phase). Vortex briefly and extract with PCI as above.
6. Precipitate RNA by adding 2.5 vols. of EtOH and incubate at -20o C at least 2-3 h. Spin down (30 min at 7000 rpm for Corex tubes or 15 min in a microfuge at max. speed for eppendorf tubes), wash pellet with 70% EtOH (5 ml for Corex or 0.75 ml for eppendorf tubes), air dry and resuspend in water (200-500 µl). The expected yield is about 50-100 µg/108 cells. This prep is suitable to be used in Northern blots or chromatographs over oligo(dT) cellulose. For more specific applications (i.e. primer extensions), further purify RNA:
7. Add 1/10 vol. of 10 x DNase I buffer (commercial) and 1/100 vol. of 1 U/µl DNase I (BRL, Amp grade). Incubate at 37o C for 30 min.
8. PCI- extract the reaction. Precipitate RNA by adding 1 vol. of 8 M LiCl to the aqueous phase. Incubate at least 5 h at -20o C.
9. Centrifuge 20 min at max speed in a microfuge, wash pellet with 70% EtOH and air dry it. Resuspend in the same volume of water and precipitate again with 1/10 vol. of 3 M NaAc, pH 5.2 and 2.5 vols. EtOH. Centrifuge and wash twice with 70% EtOH as above and resuspend in water.
10. Store at - 80o C in small aliquots. For long periods of time, store the RNA in a precipitated form at -20o C (with 1/10 vol. of 3 M NaAc, pH 5.4 and 2.5 vols EtOH).