Document Detail


Isolation of full-size mRNA from ethanol-fixed cells after cellular immunofluorescence staining and fluorescence-activated cell sorting (FACS).
MedLine Citation:
PMID:  8608737     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
C Esser; C Göttlinger; J Kremer; C Hundeiker; A Radbruch
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cytometry     Volume:  21     ISSN:  0196-4763     ISO Abbreviation:  Cytometry     Publication Date:  1995 Dec 
Date Detail:
Created Date:  1996-05-28     Completed Date:  1996-05-28     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8102328     Medline TA:  Cytometry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  382-6     Citation Subset:  IM    
Affiliation:
Medical Institute of Environmental Hygiene, University of Düsseldorf, Germany.
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MeSH Terms
Descriptor/Qualifier:
Acetic Acid
Acetic Acids
Animals
Biological Markers
Cell Separation
Cytoplasm / genetics
Ethanol
Fixatives
Flow Cytometry / methods*
Fluorescent Antibody Technique
Mice
Molecular Weight
RNA, Messenger / analysis,  isolation & purification*
Spleen / cytology
Staining and Labeling
Chemical
Reg. No./Substance:
0/Acetic Acids; 0/Biological Markers; 0/Fixatives; 0/RNA, Messenger; 64-17-5/Ethanol; 64-19-7/Acetic Acid

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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