Trizol RNA Extraction

Trizol for EC or TU tagging
TRIZOL RNA Prep. (larvae & embryos)

1. Collect ≤ 100 ml larva / embryos in 1.5 ml tube. Rinse embryos on filter and briefly spin down into tube filled with PBS then remove PBS.

2. Cover w/ 50 ml Trizol and homogenize with tissue grinder (20 – 30 seconds).

3. Add 200 ml Trizol, save at -80C until have enough samples to combine into minimum 1.0 ml prep.

4. Thaw saved Trizol samples, combine to 1.0 ml.

5. For larvae: centrifuge 10 minutes at 12,000 x g, 4 ºC, otherwise go to step 7

6. Transfer solution (insoluble pellet at bottom) to new 1.5 ml tube. Pass through 25GA needle 2X, use 3 ml syringe. CAREFUL: do not force solution through needle, only perform behind glass shield in fume hood.

7. Let sit at RT for 5 minutes

9. Add 200 ml chloroform, mix well by pipetting, let sit RT for 3 minutes

10. Centrifuge at 12,000 x g, 4 ºC, 15 minutes

11. Transfer aqueous phase to new tube (~60% starting volume)

12. Add 250 ml isopropanol and 250 ml “precipitation solution” (1.2M NaCitrate, 0.8M NaCl), mix by pipetting, let sit RT for 10 minutes

13. Centrifuge at 12,000 x g, 4 ºC, 10 minutes

14. Pour off supernatant, add 500 ml 75% ethanol and mix by swirling tube

15. Centrifuge at 7,500 x g, 4 ºC, 5 minutes

16. Pour off ethanol, air dry briefly and resuspend pellet in RNAse free water (typically      50 - 100 ml)

Required reagents (all RNAse free):
“RNA Precipitation Solution”: 1.2M NaCitrate, 0.8M NaCl in RNAse free water

75% ethanol
RNAse free water
3 ml syringes and 25GA needle