16] Extraction of high molecular weight RNA and DNA from cultured mammalian cells

Abstract

Publisher Summary This chapter describes 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. These nucleic acids are suitable for southern and northern transfers and for other applications. Extraction of RNA and DNA from mammalian cells each present different problem. 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. In the described procedure, potentially hazardous substances, such as guanidinium thiocyanate, phenol, and chloroform are avoided or minimized. The general strategy for both RNA and DNA purification is to treat with proteinase K at an early stage, such 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. The applicability of the procedures to the extraction of nucleic acids from tissues and organs has not been established.