ISOLATION OF FULL-SIZE MESSENGER-RNA FROM ETHANOL-FIXED CELLS AFTER CELLULAR IMMUNOFLUORESCENCE STAINING AND FLUORESCENCE-ACTIVATED CELL SORTING (FACS)

Preparation of intact, full-size RNA from tissues or cells requires st ringent precautions against ubiquitous and rather stable RNases. Fluor escence-activated cell sorting (FAGS) usually aims at the isolation of cells according to cell surface markers on Living cells, from which R NA can be obtained by standard protocols, The separation of cells acco rding to intracellular immunofluorescence markers, such as intranuclea r, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methano l, or formaldehyde do not inactivate RNases completely, thereby hamper ing the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting fro m cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly,and sorted by FACS. (C) 1995 Wile y-Liss, Inc.