RNA Purification

Purifing RNA is a difficult task, as RNase contamination can significantly degrade your RNA molecules. Pipette tips, tubes and all glassware should be heated before use. Aqueous solutions must be prepared with distilled, deionized and autoclaved water. In addition, the reverse transcriptase reaction will be performed in a solution containing an RNase inhibitor.

1. All cells harvested for RNA purification must be pelleted by centrifugation at 1000 rpm for 10 minutes at 4 degrees.

2. Supernatant is subsequently removed, and the pellet washed with 1 x PBS by adding 5 mL of PBS solution to the pellet and resuspending gently.

3. Re-centrifuge at 1000 rpm for another 10 minutes at 4 degrees.

4. Add 1 mL TRIZOL reagent to the pellet at the bottom of the tube.

5. Resuspend the pellet through shaking.

6. Transfer the solution to a 1.5 mL pcr tube.

7. Incubate for 5 minutes at room temperature.

8. Mix the solution by flipping the tube numerous times.

9. Add 200 microlitres of chloroform, shake the tube vigorously by hand for 15 seconds.

10. Incubate for 3 minutes at room temperature.

11. Spin at top speed at 4 degrees for 5 minutes.

12. Extract aqueous phase, which contains the RNA, and not DNA.

13. Add 0.5 mL isopropyl alcohol and leave tube at room temperature for 10 minutes.

14. Spin at top speed for 5 miutes at 4 degrees.

15. Wash pellet once with 1 mL 75% ethanol. Vortex and spin at 4 degrees for 5 minutes.

16. Remove the wash and air dry the pellet for approximately 30 minutes.

17. Resuspend the RNA in 10 uL distilled, deionized, and autoclaved H20.

18. Subsequent to this step, always have the RNA on ice to prevent degradation.

19. Dilute 1 microlitre of RNA into 1 mL of ddH20 and measure at OD 260 and OD 280 on the spectrophotometer.

20. Storage of all RNA is at -70 celcius to prevent degradation.