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Sunday, 18 November 2007

RNA isolation protocol (1)

RNA isolation by the rapid Guanidinium method
Materials

Mononuclear cell pellet isolated from 1 to 2 ml bone marrow or 5 ml whole blood
HBSS without Ca or Mg
RNA isolation solution I (25 g guanidinium thiocyanate (4 M final), 29.3 ml H2O, 1.76 ml 0.75 M sodium citrate, pH7.0 (25 mM final) , 2.64 ml 10% (w/v) N-lauroylsarcosine (0.5% final) dissolve at 65 degree celcius, store at room temperature (stable > or equal 3 months)
Do not inhale quanidinium thiocyanate and wear gloves when handling it, Do not expose guanidinium thiocyanate to acid conditions, because this may produce cyanide gas.
2 M sodium acetate, pH 4
Buffered phenol
24:1 (v/v) chloroform/isoamyl alcohol
100% and 70% (v/v) ethanol
RNA isolation solution II (10 ml RNA isolation solution I, 72 microlitre 2-mercaptoethanol (0.1 M final) Make fresh before each extraction)
DEPC-treated H2O

5 comments:

praspowt said...

1. Wash mononuclear cell pellete once with HBSS without Ca or Mg. Resuspened cells in HBSS and divide among three 1.54 ml microcentrifuge tubes. Microcentrifuge 2 min at 100 X g to pellet cells. Discard supernatant. Use immediately or store < or equal 4 months at -70 degree celcius. Use one tube to prepare RNA and the other two repeat assay.

praspowt said...

2. Add 0.5 ml RNA isolation solution I to cell pellet. Disrupt cells by repeated aspiration. Add 0.05 ml of 2 M sodium acetate. Vortex and microcentrifuge briefly at maximum speed. Add 0.5 ml buffered phenol. Vortex and microcentrifuge. Add 0.1 ml of 24:1 chloroform/isoamyl alcohol. Vortex and microcentrifuge 15 min at maximum speed 4 degree celcius.

praspowt said...

3. remove 0.4 ml of aqueous (upper) phase to a clean 1.5 ml tube that contains 0.8 ml of 100% ethanol, taking care not to disturb interface. Incubate > 1 hr at -20 degree celcius. Microcentrifuge 15 min at maximum speed, 4 degree celcius. Discard supernatant.

praspowt said...

4. Add 300 microlitre RNA isolation solution II to pellet and vortex to suspend. Microcentrifuge briefly at maximum speed. Add 600 microlitre of 100% ethanol and mix. Incubate > or equal 1 hr at -20 degree celcius. Microcentrifuge 15 min at high speed, 4 degree celcius. Discard supernatant. Wash pellet with 0.5 ml of 70% ethanol. Discard ethanol. If pellet dislodges from bottom of tube, microcentrifuge 5 min at high speed, 4 degree celcius before removing ethanol.

praspowt said...

5. Cover open tubes with Paraffin and pierce several times with a fine needle. Dry pellet briefly under vacuum or dry on bench. Store dry RNA < or equal 2 months at – 70 degree celcius or below, or dissolve in 20 microlitre DEPC-treated water if the assay will carried out immediately.