3 - Extraction of High Molecular Weight RNA and DNA from Cultured Mammalian Cells

Abstract

Publisher Summary This chapter describes the methods for the isolation of highly purified RNA and DNA in near-quantitative yield from small numbers of cultured mammalian cells and white blood cells. It is difficult to prepare both RNA and high molecular weight DNA from the same extract because each presents separate difficulties. For RNA, the most serious potential difficulty is degradation by endogenous ribonucleases. For DNA, poor yield and degradation by shearing can result if the concentration is too low. The general strategy for both RNA and DNA purification is to treat with proteinase K at an early step so that alcohol precipitation can be used to concentrate the samples and allow further processing in microcentrifuge tubes. This decreases the volume of phenol needed and also ensures near-quantitative recovery of material. Both procedures give highly purified DNA or RNA in good yield from cultured cells and from blood cells. However, the applicability of the procedures to the extraction of nucleic acids from tissues and organs is yet to be established.