I'm using white adipose tissue and brown adipose tissue for real time PCR analysis and future analysis of protein expression of PGC1a and UCP1 (using Western blot analysis). I've only had experience preparing skeletal muscle tissue samples and want to make sure I'm preparing the samples properly.
It will partially depend on how you are preparing your RNA and protein. In general, if you are using Trizol for RNA, you will have a huge fat layer after adding Trizol but before adding chloroform. The instructions from the protocol say:
- If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
The same is mostly true for lysis buffer, but because of all the detergents in the lysis buffer it is a bit less of an issue. It's sort of up to you whether you want to remove the fat layer for protein.
As a rule of thumb, use a LOT more tissue than you would think to use when comparing to muscle. Fat is somewhere about 80% Triglyceride by mass, so the amount of actual protein is going to be tiny. You'll be thinking that you're overdoing it with the tissue amount putting in, but you're not.
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