Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP).
Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.
Haukenes
G
G
University of Bergen, Norway. gunnar.haukenes@rmf.uib.no
Szilvay
A M
AM
Brokstad
K A
KA
Kanestrøm
A
A
Kalland
K H
KH
D005229:Fatty Acids, Monounsaturated; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008081:Liposomes; D008845:Microinjections; D000644:Quaternary Ammonium Compounds; D012322:RNA Precursors; D012333:RNA, Messenger / Q000032:analysis*; D014162:Transfection; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D012322:RNA Precursors; D012333:RNA, Messenger; C018009:5-bromouridine triphosphate; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane; D014544:Uridine Triphosphate