Pecinka lab, 4. 2.

2013

RNA extraction from plants using TRIZOL

HOMOGENIZATION:
1. Prepare 1.5ml Safe-lock Eppendorf tubes and add 5-6 glass-beads.
2. Add 4-6 young leaves to each tube and freeze immediately in liquid nitrogen.
Samples can be used immediately or stored for up to several monthes at -80C.
3. Cool disruptor barrels in liquid nitrogen (5 min).
4. Homogenize the tissue in a disruptor for 4 minutes at 30x amplitude
RNA extraction:
5. Immediately add 1ml of TRIZOL to the homogenized tissue
6. Vortex and incubate at RT for 10 min.
7. Centrifuge the samples at 13000 rpm for 10min at 4C to remove extracellular
material and glass beads.
8. Transfer the supernatant to new tubes.
9. Add 0.2ml of chloroform per 1ml TRIZOL
10. Shake vigorously by hand for 15 seconds.
11. Incubate at RT for 3 min.
12. Centrifuge the samples at 13000 rpm for 15min at 4C for phase separation.
13. Transfer the aqueous upper phase to new tubes (ca 50-60% of TRIZOL vol.)
14. Precipitate RNA by mixing with 0.5 ml isopropanol per 1ml TRIZOL
15. Incubate at RT for 10 minutes
16. Centrifuge the samples at 13000 rpm for 10min at 4C to obtain pellets
RNA wash:
17. Remove supernatant and wash pellets with 1ml 75% EtOH (diluted with
DEPC treated water).
18. Vortex once and centrifuge at 7500 x g for 5min at 4C
19. Discard supernatant and dry pellets for 5 min at RT (or in speed-vac)
20. Dissolve pellets in DEPC treated water
21. Incubate at 55C for 10 minutes.
Store at 80C until use.