The extraction of total RNA by the detergent and phenol method

Abstract

Successful extraction of RNA depends on the quantitative recovery of pure nucleic acids in an undegraded form. In practice, this means that a selective extraction process is required to remove all the unwanted cellular material in a manner that minimizes degradation of the RNA by hydrolysis or ribonuclease activity. The method described here relies on cell homogenization in an aqueous medium containing a strong detergent (sodium tri-isopropylnaphthalene sulfonate) and a chelating agent (sodium 4-aminosalicylate) to solubilize the cell components. An immiscible solution of phenol is then added to selectively extract hydophobic components and to denature protein. Following phase separation, the RNA is recovered by precipitation from the aqueous phase by the addition of absolute alcohol, thereby separating the RNA from small molecular weight contaminants such as carbohydrates, amino acids, and nucleotides.