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Total RNA extraction using RNeasy Kit

Reagents:

• TRIzol from Invitrogen #15596-108.
• Mini RNeasy kit from Qiagen #74134.
• Phase lock gel from Eppendorf #2302830.
• DNase I from Qiagen #79254.
• Keep TRIzol at 4C.
• Use RNase-free tubes and tips for all steps.
• Prepare 70% ethanol using DEPC-H2O and keep it at -20C.

1. Add 1.2 ml TRIzol reagent to cells/tissues (~5X10E6 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 the 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 new RNase-free tube.

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

7. Add 600 ul cold 70% ethanol to the supernatant.

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

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

10. Add 350 ul buffer RW1 to the column, centrifuge for 1 min at 14K RPM and discard the flowthrough.

11.Add 10 ul DNase I stock solution to 70 ul buffer RDD, mix by gently inverting the tube, and apply to the column. When I receive the DNase I, I prepare the solution by adding 550 ul H2O (provided in the kit) to the powder and dissolve it. I aliquot it to RNase-free tubes of 100 ul each and store at -20C. Do not vortex the solution since DNase I is susceptible to physical shear.

12. Pipette the 80 ul DNase I solution to each RNeasy column, and leave on bench for 15 min, at RT.

13. Pipette 350 ul RW1 to the column and centrifuge for 1 min at 14K RPM.

14. Wash the column with 500 ul RPE, 2 times. Spin down to remove flowthrough at 14K RPM for 1 min. Discard the flowthrough.

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

16. 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.

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

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