RNA extraction: TRIzol procedure for cell lines followed by DNase and RNeasy

• Drosophila PBS: 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, pH 7.2
• TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml)
• DNase/RNase-free water: Invitrogen cat. no. 10977.
• 75% ethanol, made with DNase/RNase-free water.
• RNase-free DNase (Qiagen cat. no. 79254.)
• RNeasy® spin columns: RNeasy midi kit (Qiagen cat. no. 75142 or 75144).

1. Pellet cells from 5-10 plates by centrifugation in 50 ml disposable polypropylene tubes (approximately 1000 x g, 5 min.).
2. Discard the supernatant. Resuspend the pellet in 5 ml Drosophila PBS; transfer to a 15 ml polypropylene tube.
3. Pellet cells again by centrifugation.
4. Remove supernatant.
5. Lyse the pellet in TRIzol reagent (0.75 ml per 5-10 x 10⁷ cells) by repetitive pipetting. For 5 plates of cells at 5x10⁶ cells/ml, use 2 ml TRIzol.
6. Incubate at room temperature for 5 minutes.
7. Add 0.267 ml chloroform per ml of TRIzol used in step 5.
8. Shake tubes vigorously for 15 seconds.
9. Incubate at room temperature for 2 minutes.
10. Aliquot samples into 1.5 ml microfuge tubes.
11. Centrifuge for 15 minutes at 4° at 12,000 x g.
12. Transfer the top (aqueous) phases to clean tubes.
13. Precipitate the RNA from the aqueous phase by adding 0.33 ml of isopropanol per ml of TRIzol used in step 5.
14. Invert tubes once to mix gently.
15. Incubate samples at room temperature for 10 minutes.
16. Centrifuge for 10 minutes at 4° at 12,000 x g.
17. Remove supernatant.
18. Wash RNA pellet once with 75% ethanol, 0.7 ml per microfuge tube.
19. Vortex briefly.
20. Centrifuge at 7,500 x g for 5 minutes at 4°.
21. Let pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely.
22. Dissolve in RNase-free water. Yield should be around 1 mg from 10 plates of cells at 5 x 10⁶ cells/ml; add enough water to give a final concentration around 2-5 mg/ml.
23. Incubate at 37° overnight to dissolve the RNA. It may be stored at -80° at this point if desired.
24. Purify up to 1 mg of RNA on an RNeasy spin column, with DNase treatment, precisely as described in Qiagen's RNeasy Midi/Maxi Handbook, supplied with the RNeasy kit; the handbook can also downloaded from http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000289. Do the optional second wash on the column. Elute twice, using 50 μl DNase/RNase-free water for each elution.
25. The yield from the column is typically 60-80%. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. If necessary, the RNA can be concentrated on a SpeedVac. Store at -80°.