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Protocol 1
total RNA extraction (acid phenol method)
(modeified from Chomczynski & Sacchi, AB 162, 156-159, 1987)


1. Reagents
  1. Trizol (LifeTech)
  2. DEPC-treated 10 mM PBS
    @Treated with diethypyrocarbonate (0.1%), for several hours, and then autoclaved.
2. Methods
  1. Under anesthesia, a rat was perfused with DEPC-treated 10 mM PBS (pH 7.4).
  2. Dissect tissues (< 100 mg)
  3. Homogenize them in 0.5 ml of Trizol with physcotron.
  4. Leave for 5 min at room temperature
  5. Add 0.2 ml of chloroform and leave for 5 min at room temperature
  6. Centrifuge for 10 min at 9000 rpm at 4Ž
  7. Carefully transfer the upper phase to a new tube
  8. Add 0.5 ml (1:1) of isopropanol and leave for 10 min at -20Ž.
  9. Centrifuge for 15 min at 15000 rpm at 4Ž.
  10. Collect the pellet and carefully add 0.4 ml of 70% ethanol.
  11. Centrifuge for 5 min at 15000 rpm at 4Ž.
  12. Collect the pellet and air-dry for 10 min at RT.
  13. Dissolve 30.0 ul of DEPC treated H2O
  14. + 20 ul DNase mix (Total 50 ul).

 

DNA mix
@5XRT10.0 ul@
@DEPCH2O7.0@
@5% BSA0.5@
@0.1MDTT1.0@
@RNase inhibitor (40 U/ul)0.5@
@DNase (10 U/ul)1.0(Pharmacia HPLC grade)
Incubate for 30 min at 37Ž
  1. Heat at 95Ž for 3 min, and cool to RT
  2. Before 30 min more, make phenol₯CHCl3₯isoamylalchohol
phenol equilibrated to pH 7.4 with Tris1 ml
Chloroform1 ml
isoamylalchohol40 ul
Vortex, and stand at RT@
  1. Add 50 ul phenol₯CHCl3₯isoamylalchohol into the DNase treated tubes and vortex.
  2. Centrifuged at 15,000 rpm for 5min at 4Ž, and collect the supernatant (40 ul).
  3. Add 40 ul of CHCl3 and vortex.
  4. Centrifuged at 15,000 rpm for 5 min at 4Ž, and collect the supernatant.
  5. Add 5 ul of 3 M AcONa buffer (pH 5.2) and 150 ul of cold 100% EtOH.
  6. Vortex well and leave at -80Ž, 15 min.
  7. Centrifuge for 15 min at 15,000 rpm and collect the pellet.
  8. Rinse carefully with 70% cold ethanol.
  9. Vaccum dry for 5 min, and resolve in 20 ul of DEPC-treated H2O or TE buffer.
    1/50 solution is used for OD checking, @RNA@1.0 OD = 40 ug/ml
    Note; When OD260/OD280<1.8, check the vigorousness of the phenol extractiion step.

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Last Updated 2005/8/8