Total RNA extraction (RNeasy plus kit)

Reagents

RNeasy plus kit contains a genomic DNA elimination column. It can replace the DNase I digestion step of regular RNeasy kit.

1. Add 1.2 ml TRIzol reagent to cells/tissues (5~10X 106 cells/ml or 50~100 mg tissue).

2. Homogenize till no debris present. For cells, lyse by pipetting. Then keep on ice.

3. Pre-centrifuge the Phase Lock Gel at 14K RPM for 3 min at RT.

4. Use sterile RNase-free tips and transfer 1 ml solution to each phase lock gel tube. add 0.2 ml chloroform. Shake it by hand to make sure the organic solution mixes well with RNA. Rotate at 4C for 20 min.

Note: Alternatively, you can transfer 1 ml solution into a RNase-free tube, add 200 ul chloroform and then proceed for following steps.

5. Spin down at 14K RPM for 15 min at 4C.

6. Transfer the aqueous phase (60% of initial TRIzol), ~600 ul to a gDNA elimination column.

Note: This step should be very easy since the lower and upper phases are separated by PLG.

7. Spin down at 14K RPM for 1 min at RT.

8. Remove the column and keep the flowthrough in the collection tube.

9. Add 600 ul cold 70% ethanol (always store at -20C) to the collection tube.

10. Pipette up and down several times to mix the solution.

11. Apply up to 700 ul of the sample to RNeasy mini column, spin down, 14K RPM for 1 min, discard the flowthrough, and apply again to load all the samples.

12. Add 700 ul buffer RW1 to the column, centrifuge for 1 min at 14K RPM and discard the flowthrough.

13. Wash the column with 500 ul RPEļ¼Œ2 times. Spin down to remove flowthrough at 14K RPM for 1 min. Discard the flowthrough.

14. Spin down to remove any carryover of RPE by centrifugation with empty collection tubes at 14K RPM for 1 min at RT.

15. Transfer the column to a new RNase-free tube, and add 30 - 50 ul RNase-free water (provided in the kit) to the center of the column. Leave on bench for 1 min, and then spin down at 14K RPM for 1 min at RT.

16. Measure RNA OD260, OD280 and concentration by nanodrop or regular spectrophotometer.

17. I usually store the RNA at -20C or immediately proceed for RT-PCR.


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