guanidine-isothiocyanate and formamide

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