Isolation of full-size mRNA 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 stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, 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, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS.
Esser
C
C
Medical Institute of Environmental Hygiene, University of Düsseldorf, Germany.
Göttlinger
C
C
Kremer
J
J
Hundeiker
C
C
Radbruch
A
A
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D015415:Biomarkers; D002469:Cell Separation; D003593:Cytoplasm / Q000235:genetics; D000431:Ethanol; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D051379:Mice; D008970:Molecular Weight; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling
D000085:Acetates; D015415:Biomarkers; D005404:Fixatives; D012333:RNA, Messenger; D000431:Ethanol; D019342:Acetic Acid