RNA Extraction Mice Adipose Tissue - (Oct/06/2009 )
I just wanted to know what's your protocol for extracting this kind of tissue? I use Trizol with an extra chloroform extraction. I haven't gotten good results from this and have never gotten an RNA pellet. Though I don't get an RNA pellet I still continue until the end and most of the time get a 260:280 ratio above 2.1 (usually around 4-6). I tested for contamination, but I always get good results for other tissue (i.e. liver). I'm just having a hard time on this one. If you can help me thanks!
In adipose tissue, you may have some contaminating fat in your sample. According to Invitrogen's protocol after homogenization, you can remove insoluble material by centrifuging at 12,000g for 10 minutes at 2-8C. In adipose tissue, there should be a layer of excess fat on top, then the supernatant with RNA, then a pellet containing other insoluble materials, like cell membranes and high molecular weight DNA. remove the fat layer, then use the supernatant for the extraction.
i forgot to mention that I do remove the fat layer. Still no pellet. I also use about .05-1 g of tissue. Does the tissue size for fat matter?
I know this topic is super old, but in case other people come across this thread.
What works very well for me is to use the standard Trizol protocol with few modifications. For every 100mg adipose tissue, add 1ml of Trizol.
The protocol states that one should move the lipid layer before adding chloroform. Do not do this! This leads to very low yield. Just continue with adding 1/5 volume chloroform. Centrifuge. Take off the aqueous fase. Add 1/5 volume chloroform to the aqueous fase. Centrifuge and continue with the standard protocol.
Following this protocol gives me nice quality RNA for RNA-seq and I usually get 50ug RNA from 500mg adipose tissue.