Extraction of total RNA from adipocytes

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we wil l describe additional steps and modifications necessary for the use of diff erent RNA isolation protocols in terms of RNA yield, RNA quality and prepar ation time. Using five times the recommended quantity of lysis buffer, incu bating the lysate at 37 degreesC. repeatedly passing the lysate through a c annula, and centrifugation to remove the lipid layer are essential addition al steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 mug total RNA from 2 x 1 0(6) cells. Preparation times were similar for all but the CsCl gradient me thod. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA, CsCl g radient ultracentrifugation is suggested for situations where DNase I diges tion is impractical.