A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization.
A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D000431:Ethanol; D019791:Guanidine; D006146:Guanidines; D007739:L Cells; D008722:Methods; D051379:Mice; D008832:Microchemistry / Q000379:methods; D006517:Murine hepatitis virus; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D051381:Rats; D012260:Ribonucleases / Q000037:antagonists & inhibitors
D006146:Guanidines; D012367:RNA, Viral; D000431:Ethanol; D012313:RNA; D012260:Ribonucleases; D019791:Guanidine