TRIzol RNA Isolation Protocol For 20 flies per tube: use gloves and eye protection Homogenization 1.

Lyse cells in 0.8ml TRIzol Reagent. 2. Remove insoluble material from the homogenate by centrifugation at 12,000 x g for 10 minutes at 4C. 3. Transfer the cleared homogenate solution to a fresh tube. Phase Separation 4. Incubate the homogenized samples for 5 minutes at 15-30C (room temperature) to permit complete dissociation of nucleoprotein complexes. 5. Add 0.2ml chloroform to tube and cap securely. 6. Shake tube vigorously by hand for 15 seconds. 7. Incubate tubes at 15-30C (room temp.) for 2-3 minutes. 8. Centrifuge samples at 11,000 x g for 15 minutes at 4C. 9. After centrifugation the mixture separates into a lower red, phenol-chloroform phase, an interphase and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase, which should be about 60% of the volume of TRIzol Reagent used in homogenization. RNA Precipitation 10. Transfer the aqueous phase to a fresh tube and save the organic phase if isolation of DNA or protein is desired. 11. Record the volume of aqueous phase added to fresh tubes. 12. Take the volume of aqueous phase and divide by 10 to determine the amount of 3M NaOAc to add to tube. 13. Take total volume (aqueous phase + 3M NaOAc) and multiply by 2 to determine the amount of 100% EtOH to add to tube. 14. Incubate samples at 15-30C (room temp) for 10 minutes. 15. Centrifuge samples at 11,000 x g for 10 minutes at 4C. (The RNA precipitate forms a gel-like pellet on the side and bottom of the tube) RNA Wash 16. Remove supernatant from tube. 17. Wash the RNA pellet once with 1ml 75% EtOH. 18. Mix sample by vortexing. 19. Centrifuge samples at 7,500 x g for 5 minutes at 4C. 20. Repeat steps 16-19 again. Redissolving the RNA 21. Remove supernatant from tube. 22. Briefly dry RNA pellet (air-dry or vacuum dry for 5-10 minutes). 23. Do not let RNA dry completely. 24. Partially dissolved RNA samples have a A260/280 ratio<1.6 (Check with spectrophotometer or nano-drop) 25. Dissolve RNA in RNase-free water (DEPC H2O) by passing the solution a few times through pipette tip. 26. Incubate for 10 minutes at 55C.