No RNA after TRIzol extraction - RNA isolation from mouse brain (Jan/05/2005 )
After dissection of mouse brain tissue I immediately homogenized it in TRIzol (hippocampus in 3 ml) and stored in -20 C for 5 days. I performed the RNA isolation according to the TRIzol protocol from Life Technologies. My water-phase wasn't very clear and after precipitation with isopropanol I couldn't see the pellets but that has happened before, so I continued. But after washing with 75% ethanol I still didn't see them. I measured my samples with the NanoDrop and obtained about 50 ng/ul RNA (total volume was 20 ul) and the 260/280 ratio was about 1,15.
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?
Are you sure that you didn't lose it during expriment? If yes, I think it is because you stored the tissue in -20. I never store tissue in -20. Why you don't do extraction immediately? If I can't do the extraction at once, I stored the tissue in -80.
I've never stored homogenized tissue before doing the TRIzol extraction, but I've done it with cells and it works fine. Brain is a tricky tissue, however, as it is rich in fat (I supposse is because of this that your aqueous phase was turbid). In my hands, the efficiency of ug RNA/mg brain is lower than with other tissues such as liver or kidney. How big was your sample? For me it helps to add 20 ug glycogen during the isopropanol precipitation. Hope it hepls!
Usually I perform the isolation immediately after dissection and the hippocampal tissue weights about 200 mg. The 260/280 values are about 1.75 and the concentration RNA about 400 ng/ul. I always dissolve the pellet in 20 ul DEPC H2O. The only thing I did different this time was freezing the TRIzol with homogenized tissue. So that's why I'm thinking that could be the reason.
Thanks anyway for your tips!
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.
Hi! MY PERSONAL EXPERIENCE IS IF YOU DO NOT WANT TO DO THE RNA EXTRACTION THE SAME DAY BETTER TO STORE THE TISSUE DIRECTLY IN LIQUID NITROGEN WITHOUT HOMOGENISATI0N BECAUSE AT -20 RNA IS NOT STABLE FOR A LONGER PERIOD OF TIME .
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.