Optimization of phenol extraction procedures for preparation of RNA from mammalian lymphoid organs

Abstract

Methods have been developed to optimize the extraction of RNA from mammalian lymphoid organs (spleen) with respect to both quantity and quality of RNA and with minimal DNA contamination. Nuclease inhibiters, including diethyl pyrocarbonate, polyvinyl suifate, and bentonite were used in the initial disruption of the tissue, which was accomplished by blender. Dounce homogenizer, or preparation of a cell suspension. Seven buffer systems, varying with respect to pH, detergent, and NaCl concentration, were used in the initial extraction with phenol, and the temperature of extraction was also varied. Protocols involving the selective use of naphthaiene 1,5-disulfenic acid and sodium dodecyl sulfate were developed to provide an initial RNA extract with minimal DNA content. Dounce homogenization, followed by separate treatment of nuclear and cytosol fractions, was found to be the most effective technique, both in terms of RNA yield (averaging 76%) and the quality of RNA recovered (as judged by gel electrophoresis). RNA from blender preparations contained larger amounts of DNA, and RNA yield was decreased to 54%. RNA extracted from spleen cell suspensions was of poor quality and gave very poor yield (27%).