[1] Isolation of RNA

Publisher Summary

This chapter discusses various methods of isolating RNA. If the purification of large initial transcripts of RNA or rare RNA intermediates undergoing processing is the goal, protocols that utilize guanidium salts to solubilize tissue and denature proteins, including ribonucleases, are the methods of choice. The preparation of RNA from subcellular fractions requires procedures for gentle disruption of cells and for inhibiting nucleases during subsequent purification of the desired molecules. The cytoplasmic components contained in the supernatant fluids are suitable for analysis of polysomes, enrichment of specific RNAs by immunoisolation of polysomes, isolation of RNA-protein complexes, and purification of cytoplasmic RNAs. The pelleted nuclei are not usually used for the isolation of RNA. The isolation of nuclei or nuclear components is usually carried out to purify nuclear RNAs or RNA processing complexes or for in vitro transcription. The efficiency of cell lysis and the quality of the nuclei can be ascertained by examination of the cell suspension before and after cell lysis in a phase-contrast microscope.