Large and Small Scale RNA Preparations from Eukaryotic Cells

A mammalian cell contains approx 10−5 μg of RNA which consists mainly of rRNA and in smaller amounts of a variety of low-mol-wt RNA species. These RNAs are of defined size and sequence. The ability to isolate clean, intact, and DNA-free RNA is a prerequisite in analyzing gene expression and cloning genes. The regulation of gene expression, e.g., the analyses of detailed function of transcription factors, promoter and enhancer sequences, and RNA synthesis and processing as well as the analyses of gene expression after transfer of genes of interest into eukaryotic cells are common areas of investigation in molecular biology. Important for the study of regulation of gene expression is the ability to isolate, analyze, and quantify RNA molecules, specifically mRNAs coding for proteins of interest. Furthermore, RNA is needed in order to copy it into double-stranded DNA for cloning and production of a cDNA library. The critical first step in the construction of a cDNA library is the efficient isolation of undegraded total RNA. The major difficulty in RNA isolation is the presence of ribonucleases found in virtually all tissues and liberated either during cell lysis or accidentally introduced in traces from other potential sources.