Materials

Qiagen RNEasy Midi Kit

Dounce homogenizers (for tissues)

Syringes and needles (for cultured cells)

70% EtOH

Sterile 15 mL conical tubes

 

Procedure

1) Before using the reagents for the first time add 10 µL of ßME per mL of Buffer RLT, and add 4 vol of EtOH to buffer RPE.

2) For tissues: Mince tissue with a scalpel and then dounce 50 - 250 µg of tissue in 2 mL of Buffer RLT. Tranfer to a sterile 15 mL conical tube. Wash the dounce with water, EtOH, and then chloroform.

For cells: Rinse a confluent 10 cm or 15 cm dish with PBS. Add 2 to 4 mL of Buffer RLT, scrape dish and and transfer to a conical tube. Vortex 10 seconds and then pass through an 18-20 G needle 5-10x.

3) Sonicate. Spin at 3K-5K rcf in a table top centrifuge at r.t. for 10 min. Pipet supernatant to new conical tube.

4) Add 1 vol (2 mL) of 70% EtOH and immediately shake vigorously. Immediately apply sample to spin column. Centrifuge 5 min at 3K-5K rcf.

5) Add 4 mL of Buffer RW1 to the column. Spin 5 min and discard flow through.

6) Add 2.5 mL of Buffer RPE to the column. Spin 2 minutes and discard flow through.

7) Repeat the the 2.5 mL wash with RPE but this time spin for 5 min. to dry the column.

8) Transfer the column to the conical collection tube. Add 250 µL of RNAse-free water for 1 minute and then spin for 3 min. Add another 250 µL of water and spin a second time. Save the flow through in a 1.5 mL eppendorf tube. Quantitate RNA by spec and by running on a gel. Store frozen.