Assessment of Cyclooxygenase RNA Expression by Northern Hybridization

Abstract

The acid guanidinium-thiocyanate phenol chloroform (AGPC) procedure is the principal method used for one-step RNA extraction and is a predominant choice for isolation of total RNA from a wide variety of biological samples. (Note 1) (1,2). This procedure permits not only isolation of RNA from large number of samples, but also recovery of total RNA from small quantities of tissue or cells. Several widely used commercial RNA preparative kits are based on this AGPC procedure. Cultured cells and tissues are homogenized in a denaturing solution containing 4 M guanidine thiocyanate. The homogenate is mixed sequentially with 2 M sodium acetate (pH 4.0), phenol, and finally chloroform/isoamyl alcohol or bromochloropropane (3). The resulting mixture is centrifuged, yielding an upper aqueous phase containing RNA, while the lower organic phase and interphase contains protein and DNA. Following isopropanol precipitation, the RNA pellet is redissolved in a denaturing solution, reprecipitated with isopropanol, and washed with 75% ethanol.