No RNA after TRIzol extraction
Posted 05 January 2005 - 04:01 AM
Does someone know what I could have done wrong? We've never stored homogenized tissue in TRIzol in the -20 C before in our lab, so could that be the reason?
Posted 05 January 2005 - 04:51 AM
Posted 05 January 2005 - 05:17 AM
Posted 05 January 2005 - 07:19 AM
badcell, on Jan 5 2005, 06:17 AM, said:
Thanks anyway for your tips!
Posted 05 January 2005 - 02:44 PM
We use a similar extraction solution called TriReagent, that says you can store the homogenate at -70 degrees. I suspect that the -20 degree storage allowed some action of RNases, despite the phenol and guanidine in Trizol.
A turbid water phase is usually the result of co-extraction of compounds similar to DNA/RNA or due to fats in suspension, these can both slow pellet formation, so I usually keep centrifuging the solution until a pellet forms. Sometimes the pellet is in the form of a gel and you have to be really careful not to discard the pellet with the supernatant and during subsequent washes.
Anyway, hope this helps.
Posted 06 January 2005 - 03:08 AM
Posted 12 January 2005 - 11:14 PM
I've extracted a lot of RNA from mouse brains and I always used Ambions kits. RNAqueous or ToTally RNA (includes phenol though). RNAqueous is easy, fast and works fine and includes no phenol. We used the RNA for qPCR and that requires good quality RNA so... We stored our brains either frozen in -80 or in RNA later (first frozen in -80 then transferred to RNAlater ICE or directly into RNAlater, both worked but RNAlater can give precipitations in the prep so I prefer RNAlater-ICE). Storing in RNAlater-ICE after freezing makes the tissue soft so you can homogenize easier and it protects the RNA from degradation.