Reagents:

Provided by Midi Kit (Qiagen, #75142, 10 samples, $90.00):

• 45 ml Buffer RLT (contains GITC, add 10ml β-Mercaptoethanol per ml RLT before use)

• 45 ml Buffer RW1 (contains GITC and ethanol)

• 11 ml Buffer RPE (concentrate, add 44 ml 100% ethanol before use)

• 10 ml RNase-free water

• 10 RNeasy Midi columns (RNA binding capacity up to 1mg each)

To be supplied by user:

• 20 cc Syringe (UW Stores, 40/box, 0056-590, $11.12)

• 14.3 M β-Mercaptoethanol (Sigma, FW=78.13, 100 ml, M-7522, $15.40)

• Ethanol (70% and 100%)

Protocol:

1. Wash cells once with PBS.

2. Lyse cells in buffer RLT (2 ml for up to 50 x 10⁶ cells).

3. Homogenize cell lysate immediately by passing 10 times through 18 gauge needle with syringe.

4. Add 1 volume (2 ml) of 70% Ethanol, mix thoroughly.

5. Transfer sample to RNeasy Midi column in 15 ml tube

6. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

7. Add 4ml of buffer RW1.

8. entrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

9. Add 2.5ml of buffer RPE.

10. Centrifuge at 3000-5000 x g for 2 minutes; discard flow-through.

11. Add 2.5ml of buffer RPE.

12. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

13. Transfer RNeasy Midi column to clean 15 ml tube.

14. Add max. 250 µl RNase-free H₂O, incubate for 1 minute.

15. Centrifuge at 3000-5000 x g for 3 minutes

16. Add max. 250 µl RNase-free H₂O, incubate for 1 minute.

17. Centrifuge at 3000-5000 x g for 3 minutes; discard column.

18. Read O.D. 260/280.

19. Give sample to CEA for Bioanalyzer run.