RNA Extraction from axolotl tissue

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Materials:

  • Trizol (Invitrogen)
  • Chloroform
  • Isopropanol
  • RNase free water
  • Potter homogenizer plus appropriate accessories (Stoessel und Pistille)
  • 5 ml syringe
  • 25 guage needle
  • Autoclaved Eppendorf tubes
  • 75% ethanol in DEPC water

    Procedure to extract total RNA:

    1. Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer
    2. Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue
    3. Homogenize the tissue by passing through a syringe with a guage needle and incubate for 5 min @ Rt
    4. Add .2 ml chloroform per ml of Trizol
    5. Vortex for 15 sec
    6. Incubate 5 min @ Rt
    7. Centrifuge for 10 min @ 4 degree and 13000 rpm
    8. Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix
    9. Mix well
    10. Incubate @ RT for 10 min
    11. Centrifuge again @ 4 degree and 13,000 rpm
    12. Remove the supernatant and wash the pellet in 75% and 100% Ethanol
    13. Air dry the pellet and dissolve in RNase free water
    14. Stores were kept in liquid nitrogen