Since these tissues are fat laden, trizol isn't working, hence I would like to stick to a kit.
We usually use Quiagen RNeasy Mini columns. They also have lipid tissue kit but normal mini columns worked fine.
Hi,
RNAeasy mini doesnot work great for human brain tissues. i have tried already.
With human and mouse brain tissue, I do use Trizol (1ML /100mg). After the Trizol protocol, I put it through High Pure RNA Isolation Kit (Roche)-DNAse I included. I put it through a column for getting rid of organics(Ph/Chl) and the genomic DNA. I get about 80-100ugs of total RNA. This mRNA works great to RT for qPCR.
When you used trizol did you try either of the following between the tissue homogenization and the phase separation with chloroform? 1. Spin at 12000 g for 10 minutes at 4 C. After the spin there is a fatty top layer that should be discarded and a clear supernatant layer which should be put into a new clean tube so you can proceed with the standard protocol. 2. Put in freezer overnight. Should lead to a similar separation with a fatty top layer that can be discarded.
In one of the protocols there was a suggestion to place tissue in liquid nitrogen prior to homogenization. It works in case of rat brain tissue I've been working with, so maybe you should try that. Also, I have good experience with Qiagen kits for RNA izolation.
Hi Deborah, I didn't go for the second step. May be I can try.
Hi Grazyna, I did do it with liq. N2 and it seems be homogenized well.
I still haven't tried TRIzol plus a kit for further purification yet.
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