Protocols

Trizol Preparation

  1. Homogenize tissue with very cold mortar and pestle or homogenizer.
  2. Transfer tissue into 1.7mL Eppendorf tube or 25mL Falcon tube.
  3. Add 10μL TRIZOL / mg tissue, vortex thoroughly.
  4. Run tissue/TRIZOL mixture through a 20 gauge needle several times to complete homogenization.
  5. Let stand at room temperature for 5 minutes.
  6. Add 20μL chloroform /100uL TRIZOL, vortex for 15 seconds, and leave at room temperature for 3 minutes.
  7. Centrifuge samples at maximum rpm for 15 minutes at 4°C.
  8. Transfer aqueous phase to a fresh tube (the use of Eppendorf PhaseLock Gel greatly decreases organic phase contamination at this step).
  9. Add 50μL isopropyl alcohol /100uL TRIZOL and incubate at room temperature for 10 minutes.
  10. Centrifuge samples at maximum rpm for 10 minutes at 4°C.
  11. Remove supernatant.
  12. Wash RNA pellet with 80% ethanol and vortex.
  13. Centrifuge samples at 7,500g for 5 minutes at 4°C.
  14. Remove supernatant.
  15. Reconstitute pellet in 20μL of RNase free water.

Lysis Buffer

  • 50 mM Tris pH 8.0
  • 50 mM EDTA
  • 25 mM Sucrose
  • 100 mM NaCl
  • 1% SDS

Low TE pH 8.0

  • 10 mM Tris pH 8.0
  • 0.1 mM EDTA pH 8.0

RNAlater

  • 25 mM Sodium Citrate
  • 10 mM EDTA
  • 70 g ammonium sulfate/100 ml solution, pH 5.2

(Information taken from patent information: US Patent 6,204,375)