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RNA Isolation

Make sure all tubes, reagents, and equipment have been treated to remove residual RNase contamination before beginning RNA prep. Make sure the TRIZOL Reagent is at room temp before starting the procedure. Wear gloves and eye protection when working with TRIZOL reagent.

  1. Grind tissue with mortar and pestle with liquid nitrogen.
  2. For each 100mg of tissue, use 1.5ml of TRIZOL reagent to a maximum of 1 gram (use only glass or polypropylene tubes with reagent). Add the ground tissue to an appropriate amount of TRIZOL and mix thoroughly by hand for at least two minutes.
  3. Centrifuge at 12,000xg for 10 min at 4C
  4. Transfer supernatant to a new tube and incubate for 5 min at room temp.
  5. Add 0.2ml chloroform per 1ml TRIZOL and shake by hand for 20 seconds.
  6. Incubate at room temp for 2-3 minutes.
  7. Centrifuge at 12,000xg for 15 min at 4C
  8. Transfer the colorless upper aqueous phase containing the RNA to a new tube and discard organic tissue.
  9. Add 0.5 ml isopropanol for each 1ml of TRIZOL used to precipitate RNA
  10. Incubate samples at room temp for 10 minutes.
  11. Centrifuge at no more than 12,000xg for 10 minutes at 10 minutes at 4C.
  12. Wash pellet in 75% ethanol and air dry