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 will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.
Janke
J
J
Franz-Volhard-Klinik, Universitätsklinikum Charité, Humboldt Universität Berlin, Germany.
Engeli
S
S
Gorzelniak
K
K
Sharma
A M
AM
D017667:Adipocytes / Q000737:chemistry* / Q000166:cytology; D002478:Cells, Cultured; D002586:Cesium; D002712:Chlorides; D002725:Chloroform; D004202:Disinfectants; D005821:Genetic Techniques; D006801:Humans; D019800:Phenol; D012313:RNA / Q000302:isolation & purification*; D014461:Ultracentrifugation
D002712:Chlorides; D004202:Disinfectants; D002586:Cesium; D019800:Phenol; D012313:RNA; D002725:Chloroform; C028019:cesium chloride