This protocol uses the Oligotex mRNA Midi Kit (Qiagen).
1. Mix cell lysate (600 µl in OCL buffer for 1-5x106 cells)
or 600 µl (1 mg) total RNA with 600 µl 2X binding buffer.
2. Add 35 µl Qiagen Oligotex-dT resin.
3. Heat 3 minutes at 70 °C to denature the RNA.
4. Cool to room temperature 10 minutes to allow annealing of mRNA to resin.
5. Pellet the resin containing bound mRNA by spinning for 2 minutes in a microfuge.
6. Resuspend the resin in 600 µl of wash buffer by vortexing vigorously.
7. Pellet the resin by centifuging in a microfuge at the maximum speed.
8. Resuspend the resin in 200 µl of QL1 wash buffer.
9. Add 800 µl of ODB buffer.
10. Pellet the resin and then resuspend in 600 µl of OW1 buffer.
11. Transfer the resin to a Qiagen spin column.
10. Centrifuge at maximum speed for 30 seconds, discard flow-through.
11. Wash with 600 µl of OW2, discard flow-through.
12. Repeat step 11 once.
13. Elute with 33 µl 80 °C elution buffer OEB.
14. Repeat step 13 twice.
15. Combine the three 33 µl mRNA fractions.
16. Precipitate with ethanol if necessary.