Protocol for RNA Isolation From Tissue Samples
(Chomczynski and Sacci, BioAnalytical Chemistry 162: 156-159, 1987)

1. Homogenize tissue with Solution D (1ml solution D/100mg tissue).
2. Add 2M Sodium Acetate (0.1ml/100mg tissue).
3. Mix by inversion.
4. Add Phenol equilibrated to ~pH 4.5 (1ml/100mg tissue).
5. Mix by inversion.
6. Add Chloroform (0.2ml/100mg tissue).
7. Mix by inversion.
8. Vortex vigorously for 10 seconds.
9. Cool on ice.
10. Centrifuge at 10,000xg for 20 minutes at 4^oC.
11. Transfer aqueous phase to a fresh tube.
12. Add isopropanol (1ml isopropanol/100mg tissue).
13. Vortex briefly.
14. Incubate at -20^oC for at least 1 hour.
15. Centrifuge at 10,000xg for 20 minutes at 4^oC.
16. Discard supernatant.
17. Dissolve pellet in Solution D (0.3ml/100mg tissue).
18. Add Isopropanol to precipitate (0.3ml/100mg tissue).
19. Incubate at -20^oC for at least 1 hour.
20. Centrifuge at 10,000xg for 20 minutes at 4^oC.
21. Discard supernatant.
22. Wash pellet with 70% ethanol.
23. Centrifuge at 10,000xg for 10 minutes at 4^oC.
24. Air-dry the pellet for 10 minutes.
25. Dissolve pellet in desired buffer (50µl/100mg tissue).

Denaturing solution - Solution D (add 360µl beta-mercaptonethanol to 50ml immediately before use): 4 M Guanidine (isothiocyanate), 25 mM Sodium Citrate, 0.5% SDS.