Total RNA extraction
*This protocol was successful for extracting RNA for the Suillus brevipes and Wilcoxina mikolei transcriptomes. Tissue was grown in agar plates with a layer of cellophane and mycelium harvested, flash frozen, and stored at -80C (agar plug was discarded). Use filtered tips.
(no more than 8 samples at a time)
1. Add the following to a 2 ml screw top (ice, hood):
5mm depth of 0.5mm zirconia/silica beads (c.a. 200mg)* [mark a tube and use that volume]
1ml Trizol (Invitrogen 15596-026)
frozen mycelium (appx 1cm x 2cm mycelium grown on cellophane)
2. Beadbeat at max speed for 30sec twice (Mini Bead Beater, Biospec). Cool tubes in between beats.
3. Gentle shake for 5min at room temperature for nucleosome removal. (rotator on cDNA bench)
4. Add 0.2ml chloroform and beadbeat at max speed for 15sec. [make sure not to pull water on top of chloroform].
5. Gentle shake for 2min at room temperature.
6. Centrifuge 12,000xg for 15min at 4C, collect supernatant (hood!; only top part – no junk!; there’s ~600 ul, but use 200 tips to control for supernatant only; discard liquid carefully in chloroform waste bottle so beads are kept in tube)
7. Add 0.5ml isopropanol and mix well. (Regular nuclease-free tubes are better than low-adhesive tubes when doing alcohol precipitation).
8. Gentle shake for 10 min at room temperature. (rotator).
9. Centrifuge 12,000xg for 10min at 4ºC (cold room) discard supernatant. Use RNA bench – new gloves, wipe the tubes, gloves and bench with RNase AWAY before discarding supernatant.
10. Wash pellet with 1ml 75 (or 70)% ethanol (can be cold), vortex, and spin 7,500xg 5 min at 4ºC. The pellet is not always visible, so be consistent with the tube position on the centrifuge. RNase AWAY tubes again.
11. Dry pellet (don’t over dry! 5-10 min) - basically discard supernatant without disturbing the pellet.
12. Add 100μL water – good for RNeasy kit, but TE is better for long term storage. Mix, so the pellet is in solution. Flick if needed. Quick spin.Add 1 ul RNase out.
13. Quantify total RNA (use 1/10 dilution for Qubit).
14. Purify total RNA using the RNeasy Mini Protocol for RNA Cleanup, elute in 30µL RNase free TE (or 35 – pass elution through the column twice for more and more concentrated RNA); do the DNase step, do the optimal step 6; use 1.5 low retention tubes in step 7; when centrifuging, close transparent lid (and mark it!) and cut-off pink lid (this will be the final RNA tube); do a LiCl precipitation (add same volume of 4M LiCl, store at -20C overnight, precipitate in the morning (just like an ethanol precipitation)).
15. Quantify (step 13)
Use nanodrop to check the quality of RNA – Ideally samples should be between 1.8 and 2.2.; parafilm the lid to make sure tube is properly closed and store at -80.
16. Send to Bioanalyzer – prep tubes with 2 ul 1/10 dilutions and adjust [RNA] to 5ng tops:
V(H2O) = (2ul x [clean RNA])/5ulmg
Quantification