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Reagents and kits 1. Ficoll (Amersham Biosciences) 2. Ice-cold phosphate buffer (PBS), pH 7.43. Lysis buffer: 5mol / L guanidine monothiocyanate, 10mmol / L ethylenediaminetetraacetic acid (EDTA), 50mm
Instruments, consumables 1. 30m1 Corex tube, pickled and siliconized 2. Pre-cooled centrifuge and bucket rotor
Experimental Step 1. Collect fresh human blood and immediately isolate leukocytes with Ficoll.
2. Wash the peripheral blood lymphocytes 3 times with ice cold PBS.
3. Collect peripheral blood lymphocytes in a 50ml plastic centrifuge tube, and add 7ml of lysis buffer (which can dissolve up to 5 x 108 peripheral blood lymphocytes), then vortex vigorously to lyse the cells.
4. Add 7 volumes (49 ml) of 4 mol / L lithium chloride and incubate at 4 ° C for 15 to 20 hours (overnight).
5. Transfer the suspension to a 30ml Corex tube, centrifuge at 4 ° C for 2 hours in a bucket-type rotor, 6500r / min.
6. Discard the supernatant and wipe the test tube opening with Kimwipe. Resuspend with 3mol / L lithium chloride (approximately 15ml) and collect the precipitate. Centrifuge the resuspended precipitate at 6500r / min for 1 hour.
7. Discard the supernatant and dissolve the precipitate with 2ml RNA lysis solution. At -20 ° C, freeze the suspension thoroughly.
8. Resuspend the suspension and vortex every 10 minutes for 20 seconds for a total of 45 minutes.
9. Extract once with equal volume of phenol and once with equal volume of chloroform.
10. Add 1/10 volume of 3 mol / L sodium acetate, pH 4.8 and 2 volumes of ethanol at 20 ° C. Mix the solution thoroughly and incubate at -20 ° C overnight.
11. Centrifuge the RNA in the bucket rotor, 12000r / min for 30 minutes. Treat the water with 0.2ml DEPC to resuspend the pellet. Transfer the dissolved DNA to a 1.5 ml microcentrifuge tube, add 1/10 volume of 3 mol / L sodium acetate, pH 4.8 and 2 volumes of -20 ° C ethanol to re-precipitate. And save the RNA in the form of ethanol precipitate until ready to use.
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