实验步骤

1. Ground sample with liquid nitrogen.

2. Weight 10-50 mg ground sample into a tube contains 400 ul CPL Buffer. Mix the sample throughly by vortexing the tube 20 seconds.

3. Add 400 ul chloroform and mix throughly by vortexing for 20 seconds.

4. Centrifuge at 13,000 x g for 5 minutes.

5. Transfer 300 ul of upper aqueous phase into a new 1.5 ml microtube. Add 300 ul of isoproanol to precipitate nucleic acid.

6. Centrifuge at 13,000 x g for 5 minutes. Remove the supernatant and dissolve RNA by 300 ul DEPC Water.

7. Add equal volume of the RB Buffer followed by 325 ul absolute ethanol (96-100%). Mix the sample thoroughly by vortexing.

8. Load 600 ul of the sample into the HiBind RNA column. Centrifuge at 10,000 x g for 1 minutes. Discard the flow-through and re-use the collection tube.

9. Apply remaining sample to the HiBind RNA column by repeating step 8. Discard the flow-through and collection tube.

10. Place the RNA column into a new collection tube. Add 500 ul RNA Wash Buffer I into the column. Centrifuge at 10,000 x g for 1 minutes. Discard the flow-through and re-use the collection tube.

11. Add 500 ul RNA Wash Buffer II into the column . Centrifuge at 10,000 x g for 1 minutes. Discard the flow-through and re-use the collection tube.

12. Add 500 ul RNA Wash Buffer II into the column . Centrifuge at 10,000 x g for 1 minutes. Discard the flow-through and re-use the collection tube.

13. Centrifuge the empty column for 2 minutes to dry the column.

14. Place the column into a new 1.5 ml centrifuge tube. Add 100 ul DEPC Water into the center of the membrane in the column. Incubate at RT for 2 minutes.

15. Centrifuge at maximum speed (>13,000 x g) for 1 minutes to elute RNA.