Trizol Isolation of RNA
1. Homogenize tissue in trizol:
brain 1:10 and heart 1:20
2. Centrifuge at 5k for 10min at 4C
3. Remove supernatant (clear layer) and leave pellet and to top fat layer.
4. sit at RT for 5 min
5. Add 0.2 ml chloroform per 1 ml trizol
6. Shake by hand for 15 seconds
7. Incubate at RT for 3 minutes
8. Centrifuge 15 min @ 5K at 4C
9. Pull off top clear supernatant and keep (about 60% of homogenization volume)
10. Transfer to fresh tube
11. Add to supernatant 0.25 isopropyl per 1 ml trizol and 0.25 ml of 0.8M sodium
citrate, 1.2M NaCl (2.4g of citrate and 0.7 g of NaCl in 10 ml) per 1 ml of trizol (this is
for muscle tissue)
12. Incubate at RT for 10minutes and centrifuge 5K for 10 minutes
13. Remove supernatant.
14. Wash the RNA pellet once with 75% EtOH (adding 1ml for every 1ml of Trizol
used)
15. Vortex and centrifuge at no more than 7.5K g for 5 minutes at 4C