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HOME > Protocols > Molecular Biology > RNA > Protocol for RNA Isolation from Tissue Samples

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 4oC.
  11. Transfer aqueous phase to a fresh tube.
  12. Add isopropanol (1ml isopropanol/100mg tissue).
  13. Vortex briefly.
  14. Incubate at -20oC for at least 1 hour.
  15. Centrifuge at 10,000xg for 20 minutes at 4oC.
  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 -20oC for at least 1 hour.
  20. Centrifuge at 10,000xg for 20 minutes at 4oC.
  21. Discard supernatant.
  22. Wash pellet with 70% ethanol.
  23. Centrifuge at 10,000xg for 10 minutes at 4oC.
  24. Air-dry the pellet for 10 minutes.
  25. Dissolve pellet in desired buffer (50Ál/100mg tissue).

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


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