RNA isolation

RNA isolation
(SV Total RNA Isolation System, Promega)
(V. Orgogozo, 2007)

1. With sterile equipment, dissect flies in cold PBS and put tissues in a 1.5mL tube on ice containing 175uL of SV RNA Lysis Buffer. Put about 20 pairs of testis or 20 pairs of ring gland-brain complexes per tube. Store at -20C for several days-months.
2. Homogenize completely (very important, to inactivate all the RNAses present in the tissues) with sterile blue pestles that have been cleaned with RNAse away ( ).
3. Add 350uL SV RNA Dilution Buffer and mix by inverting the tube 3-4 times.
4. Incubate at 70C for 3min (but no longer otherwise RNA might break down).
5. Centrifuge for 10min at 13,000rpm.
5. Transfer the clear lysate to a clean 1.5mL tube.
6. Add 200uL of ethanol 95% and mix by pipetting 3-4 times.
7. Transfer to the Spin Column Assembly and centrifuge for 1min at 13,000rpm.
8. Pour the flow-through. Add 600uL of SV RNA Wash Solution and centrifuge for 1min at 13,000rpm.
9. Pour the flow-through. Add 50uL of DNase mix (40uL Yellow Core Buffer + 5uL MnCl2 + 5uL DNAse I (aliquots in Chantal's brown box at -20C)).
10. Incubate at room temperature for 15min.
11. Add 200uL SV DNase Stop Solution and centrifuge for 1min at 13,000rpm.
12. Add 600uL of SV RNA Wash Solution and centrifuge for 1min at 13,000rpm.
13. Pour the flow-through. Add 250uL of SV RNA Wash Solution and centrifuge for 2min at 13,000rpm.
14. Transfer the column to a new 1.5mL tube and add 100uL of nuclease-free water to the center of the filter. Wait 1min and centrifuge at 13,000rpm for 1min.
15. Store at -80C.