THE PREPARATION AND APPLICATIONS OF CYTOPLASMIC EXTRACTS FROM MAMMALIAN CELLS FOR STUDYING ASPECTS OF MRNA DECAY

HeLa 5100 cytoplasmic extracts have been shown to effectively recapitulate many aspects of mRNA decay. Given their flexibility and the variety of applications readily amenable to extracts, the use of such systems to probe questions relating to the field of RNA turnover has steadily increased over time. Cytoplasmic extract systems have contributed greatly to the field of RNA decay by allowing valuable insight into RNA-protein interactions involving both the decay machinery and stability/instability factors. A significant advantage of these systems is the ability to assess the behaviors of several transcripts within an identical static environment, reducing errors within experimental replications. The impact of the cytoplasmic extract/in vitro RNA decay technology may be further advanced through manipulations of the extract conditions or the environment of the cells from which it is made. For instance, an extract may be produced from cells after depletion of a specific factor by RNAi, giving insight into the role of that factor in a particular process. The goals of this chapter are threefold. First, we will familiarize the reader with the process of producing high-quality, reliable HeLa-Cell cytoplasmic extracts. Second, a method for the standardization of independent extracts is described in detail to allow for dependable extract-to-extract comparisons. Finally, the use and application of cytoplasmic extracts with regard to assaying several aspects of mRNA turnover are presented. Collectively these procedures represent an important tool for the mechanistic analysis of RNA decay in mammalian cells.