Preparation of cytoplasmic RNA from tissue culture cells.
The protocol described in this unit is a fast and streamlined procedure for preparing total cytoplasmic RNA from many cultures simultaneously for nuclease protection analysis. It is scaled for small cultures--1 to 2 dishes of adherent cells or 10 to 20 ml of a suspension culture. The procedure works well for many cell types. If full-length RNA is required, ribonuclease inhibitors should be added to the lysis buffer (as described in this unit) or the guanidinium isothiocyanate method should be used. Finally, if RNA is isolated from transiently transfected cells, a is provided for the treatment of RNA with deoxyribonuclease to remove transfected DNA. This modification is especially critical if the RNA is to be assayed by nuclease protection using uniformly labeled probes.
Gilman
Michael
M
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
D000818:Animals; D002478:Cells, Cultured / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification; D005057:Eukaryotic Cells / Q000737:chemistry; D007202:Indicators and Reagents; D011387:Prokaryotic Cells / Q000737:chemistry; D012313:RNA / Q000302:isolation & purification*
D007202:Indicators and Reagents; D012313:RNA; D004247:DNA