Isolating RNA from clinical samples with Catrimox-14 and lithium chloride.
RNA is a highly informative molecule that has great potential as a target for diagnostic studies. This potential can be reached only when reliable methods for isolating RNA are available in the clinical environment. Cationic surfactants lyse cells and precipitate nucleic acids. We have described a novel cationic surfactant (tetradecyltrimethylammonium oxalate, Catrimox-14), which is particularly effective in precipitating RNA from cells and which can be applied to clinical specimens. We examine the utility of a method of recovering RNA from the surfactant-nucleic acid precipitate, in which 2 M lithium chloride is used to extract the DNA and surfactant from the precipitate; RNA (being insoluble in lithium chloride solution) remains in the pellet. The yield of RNA from peripheral blood mononuclear cells by the Catrimox-LiCl method we describe was the same yield by a conventional method using guanidine thiocyanate, phenol, and chloroform (GPC). The quality of the RNA, judged by agarose gel electrophoresis, A260/280 ratio and its ability to serve as a target for reverse transcription and PCR, was the same. RNA was isolated and amplified from blood stored for at least 2 weeks in Catrimox solution at room temperature. RNA was also easily isolated with the Catrimox-LiCl method in good yield from frozen sections of mouse liver, spleen, kidney and brain, and from core biopsies of liver and kidney. RNA isolated from needle aspirates of liver, spleen, kidney, pancreas, and brain was easily amplified by RT-PCR. The Catrimox-LiCl method is simple and does not call for the use of corrosive reagents. The Catrimox-LiCl method removes 98% of the DNA. We conclude that the Catrimox-LiCl method is suitable for use in clinical applications of RNA-based diagnosis.
Macfarlane
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DE
Department of Medicine, University of Iowa College of Medicine, Iowa City, USA.
Dahle
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CE
D000818:Animals; D001707:Biopsy, Needle; D001774:Blood Chemical Analysis; D016616:Cryoultramicrotomy; D005260:Female; D007668:Kidney / Q000737:chemistry; D018021:Lithium Chloride / Q000737:chemistry*; D008099:Liver / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012313:RNA / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000737:chemistry*; D050337:Trimethyl Ammonium Compounds
D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; D050337:Trimethyl Ammonium Compounds; D012313:RNA; D018021:Lithium Chloride; C009410:tetradecyltrimethylammonium