This chapter discusses the conditions required to isolate high molecular weight cellular RNA suitable for exogenous expression. The chapter also describes several protocols (gel electrophoresis, Northern blot analysis, and RNase protection) useful for the initial characterization of purified RNA. RNA is highly susceptible to both enzymatic and chemical degradation; chemical degradation of RNA can occur at all stages of RNA purification, handling, and storage. Total cellular RNA can be separated from other cellular constituents by selective precipitation of RNA or by density gradient centrifugation. The two protocols— guanidinium thiocyanate/cesium chloride method and lithium chloride/urea method—give good yields of high molecular weight RNA that is translatable in oocytes. Northern blot analysis using RNAs isolated from various tissues and developmental stages can give an indication to the source that would provide the most robust signal on injection of RNA into oocytes.
ASJC Scopus subject areas
- Molecular Biology