RNA isolation

With reference to Jess's post on 28 October, I will add on about how to isolate RNA from cells.

RNA is isolated from the cells using NucleoSpin RNA Isolation kit.

NucleoSpin RNA Isolation (Note: Centrifuge at room temperature)

1. Prepare DNase reaction mixture in a sterile 1.5ml microcentrifuge tube; for each isolation/reaction add 10ml reconstituted rDNase to 90ml Reaction Buffer for rDNase.

2. Add 350ml Buffer RA1 and 3.5ml b-mercapethanol to the cell pellet and vortex vigorously to lyse the cells. Note: Addition of b-mercapethanol should be done in the hood.

3. Place NucleoSpin Filter columns (violet ring) in a collection tube and filter the lysate through NucleoSpinâ Filter columns (violet ring) to reduce viscosity and clear the lysate, at 11 000 x g for 1 minute.

4. Transfer the flow-through to a 1.5ml micocentrifuge tube and add 350ml of 70% ethanol. Mix by vortexing (2 X 5 seconds).

5. Pipet the lysate up and down 2-3 times and load the lysate to the NucleoSpin RNA II columns (light blue ring) and centrifuge at 11 000 x g for 30 seconds.

6. Add 350ml of Membrane Desalting Buffer (MDB) and centrifuge at 11 000 x g for 1 minute to dry the membrane (desalt silica membrane – desalting step make rDNase digest much more effectively). Note: Discard the flow-through.

7. Add 95ml DNase reaction mixture prepared in step 1 directly onto the center of silica membrane of the column and incubate at room temperature for 15 minutes.

8. (1^st Washing) Add 200ml Buffer RA2 to the column and centrifuge at 11 000 x g for 30 seconds (Buffer RA2 inactivates the rDnase). Discard the flow-through.

9. (2^nd Washing) Add 600ml Buffer RA3 to the column and centrifuge at 11 000 x g for 30 seconds. Discard the flow-through.

10. (3^rd Washing) Add 250ml Buffer RA3 to the column and centrifuge at 11 000 x g for 2 minutes to dry the silica membrane completely. Then place the column in a nuclease-free collection tube.

11. Elute the RNA in 30ml RNase-free water and centrifuge at 11 000 x g for 1 minute.

12. Store RNA in -80^oC.

Li Yinliang Alex 0704894E

TG02 Group 8

2 November 2009