Labeling of RNA Transcripts of Eukaryotic Cells in Culture with BrUTP Using a Liposome Trans-fection Reagent (DOTAP~R)

摘要

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~R). Within minutes, DOTAP mediatesuptake of BrUTP both at 37°and4°C. This is followedby 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.