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Star Republic: Guide for Biologists

RNA purification --- Qiagen Rneasy

This protocol uses the Rneasy Kit from Qiagen. RLT, RW1, and PRE buffers are included in the kit.

1. Homogenize cells (5-10 x 106 cells) or tissue (~ 50 mg) in 600 Ál RLT/▀-mercaptoethanol.

2. Centrifuge for 12 minutes at maximum speed in a microfuge. Transfer supernatant fluid, but not surface layer, into new tube.

3. Add 600 Ál 70% ethanol, mix.

4. Apply up to 700 Ál of sample to an RNeasy mini column and allow to pass through under vacuum or by centrifugation for 15 seconds at 12,000 rpm. Load the remaining sample and allow to pass through.

5. Run 700 Ál RW1 through the mini column, under vacuum or 15 seconds at 12,000 rpm.

6. Run 500 Ál RPE/ethanol through the column, under vacuum or 15 seconds at 12,000 rpm.

7. Repeat the wash step 6.

8. If using centrifugation, change the collection tube, and centrifuge at maximum speed for 2 minutes or until the column is dry.

9. Transfer the column to a new collection tube, add 30-50 Ál RNase-free ddH2O directly to the filter of the column, incubate for 5-10 minutes, and spin for 1 minute at 12,000 rpm.

10. Dilute an aliquot (1:20 to 1:100) to determine the RNA concentration using a spectrophotometer.