lithium-chloride and tetradecyltrimethylammonium

Topics: DNA; Humans; Lithium Chloride; Molecular Biology; Quaternary Ammonium Compounds; RNA; RNA, Bacterial; RNA, Plant; Surface-Active Agents; Trimethyl Ammonium Compounds

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.

Topics: Animals; Biopsy, Needle; Blood Chemical Analysis; Cryoultramicrotomy; Female; Kidney; Lithium Chloride; Liver; Mice; Mice, Inbred BALB C; Monocytes; Polymerase Chain Reaction; Quaternary Ammonium Compounds; RNA; Surface-Active Agents; Trimethyl Ammonium Compounds

Traditional RNA isolation methods use chaotropic agents and anionic detergents to lyse cells and solubilize nucleic acids. In contrast, the cationic surfactant, Catrimox-14, lyses cells and simultaneously precipitates RNA, thereby protecting it from RNases. We describe and compare four methods for extracting RNA from cultured cells that differ in the technique used to extract the RNA from the precipitate. The first uses a high-salt solution (guanidinium isothiocyanate). In the second, the RNA is extracted with a polar solvent (formamide). The third involves conversion of the RNA to the sodium salt by treatment of the precipitate in situ with sodium acetate in ethanol. The fourth uses 2 M lithium chloride to convert the RNA in the pellet to the lithium salt in situ. We applied these methods to human leukemia cells growing in culture and each method resulted in excellent yields of RNA (typically 23 micrograms/million K562 cells, 13 micrograms/million HL-60 cells) over a wide range of cell concentrations (1 x 10(5) - 3 x 10(7)/ml) and of good to excellent quality as judged by agarose electrophoresis and UV absorbance data (OD260/280 1.90-2.05). The advantages and limitations of each method are discussed.

Topics: Acetates; Acetic Acid; Cations; Detergents; Electrophoresis, Agar Gel; Formamides; Guanidines; Humans; Isothiocyanates; Leukemia, Erythroblastic, Acute; Leukemia, Promyelocytic, Acute; Lithium Chloride; Quaternary Ammonium Compounds; RNA, Neoplasm; Trimethyl Ammonium Compounds; Tumor Cells, Cultured