While working with RNA:
Wipe down area with RNAzap.
Have dedicated “RNAse free” tips, solutions, etc.
Use filter tips.
1. Collect worms (collect from 5 small plates that are close to starved out)
2. Wash worms with M9 (2 to 3 times)
3. Freeze worms at -80. (You can stop here.)
4. Grind worms with chilled mortar. To get pellet out of 1.5mL tube, use pliers to break the tube and free the pellet. Chill to -80. (Do not thaw worms while grinding!). Liquid N2 may be helpful!
5. Add 9 times Trizol (Invitrogen, stored in Flammable Cabinet 3) to mortar and wait for worms to thaw. Transfer to a 1.5mL tube. Vortex.
Ex) 0.9ml trizol to 0.1ml worm
6. Leave it 10-15 min at room temp.
7. Add 0.2 ml of chloroform per 1 ml of TRIZOL and vortex.
8. Incubate them at 15 to 30°C for 2 to 3 minutes
9. Centrifuge the samples at no more than 12,000 × g for 15 minutes at 2 to 8C (14,000rpm at room temp was OK in my case).
10. Transfer the aqueous phase to a fresh tube. For RNA prep, add phenol/chloroform (stored in deli fridge) and vortex and centrifuge again. Transfer the aqueous phase (top) to a fresh tube.
11. Add equal amount of isopropanol.
12. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 × g for 10 minutes at 2 to 8°C (14,000rpm at room temp was OK in my case). Remove isopropanol and look for RNA pellet at the bottom of the tube.
13. Rinse RNA with with 75% ethanol (RNAse free).
14. Dissolve RNA in water or TE (Do not dry RNA up completely or it never dissolves!). Use 200uL TE if isolating worms from 5 plates.
15. Run out 1% of your RNA on a gel to check that it was not degraded during isolation.
For cDNA synthesis, I use SuperscriptIII (Invitrogen) 180800051, based on its protocol.
20ul scale may be enough for normal RT-PCR, and I usually use 2-4ul as template.
EDIT 10/17/2016 (Becky): Clarified step 10 (previously, 10. Transfer the aqueous phase to a fresh tube. For RNA prep, add phenol/chloroform (stored in deli fridge) and vortex again. Remove the aqueous phase (top).)