Method

1. Rinse the tissue culture plates of transfected COS-7 cells three times with ice-cold PBS lacking calcium and magnesium ions. Keep plates on a bed of ice between rinses.

2. Add 10 ml of ice-cold PBS to each plate. Place the plate on the bed of ice, and gently scrape the cells off the plate.

3. Transfer the cell suspensions to chilled 15-ml polystyrene tubes.

4. Recover the cells by centrifugation at 300g (1200 rpm in a Sorvall H1000B rotor) for 8 minutes at 4°C.

5. Decant as much of the supernatant as possible. Remove the residual supernatant with a pipette.

6. Resuspend the cell pellet from 1 x 106 to 2 x 106 COS cells in 300 pl of TKM buffer and store the suspension on ice for 5 minutes.

7. Add 15 pl of 10% Triton X-100 or 4%-Nonidet P-40 and store the cell suspension on ice for a further 5 minutes.

8. Recover the nuclei by centrifugation at 500g (1500 rpm in a Sorvall H1000B rotor) for 5 minutes at 4°C.

9. Transfer the supernatant to a chilled 1.5-ml microfuge tube.

IMPORTANT Use extreme care when removing the supernatant. Do not touch the nuclear pellet. If the nuclear membranes burst, the sample will become too viscous to pipette due to the contamination with genomic DNA.

10. Add 20 pl of 5% SDS and 300 pl of phenol. Vortex the mixture vigorously and separate the organic and aqueous phases by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.

11. Transfer the aqueous layer to a 1.5-ml microfuge tube containing 300 pl of phenol:chloroform. Vortex the suspension vigorously and then separate the organic and aqueous phases by centrifugation at maximum speed for 3 minutes at room temperature in a microfuge.

12. Transfer the aqueous (upper) layer to a chilled 1.5-ml microfuge tube containing 12 pl of 5 M NaCl and 750 pl of ethanol. Allow the RNA to precipitate for 2-3 hours at -20°C or for 30 minutes at -80°C.

13. Recover the RNA by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge.

14. Discard the supernatant and wash the pellet with 70% ethanol. Dry the pellet in air and redissolve it in 20 pl of DEPC-treated H2O.

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