Sokoloski Kevin J, Wilusz Jeffrey, Wilusz Carol J
Department of Microbiology, Immunology and Pathology, Colorado State University, Ft. Collins, Colorado, USA.
Methods Enzymol. 2008;448:139-63. doi: 10.1016/S0076-6879(08)02608-6.
HeLa S100 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.
已证明HeLa S100细胞质提取物能有效重现mRNA降解的许多方面。鉴于其灵活性以及提取物易于应用的多样性，随着时间的推移，使用此类系统来探究与RNA周转领域相关问题的情况稳步增加。细胞质提取物系统通过使人们能够深入了解涉及降解机制以及稳定性/不稳定性因子的RNA-蛋白质相互作用，为RNA降解领域做出了巨大贡献。这些系统的一个显著优势是能够在相同的静态环境中评估多个转录本的行为，减少实验重复中的误差。通过操纵提取物条件或制备提取物所用细胞的环境，细胞质提取物/体外RNA降解技术的影响可能会进一步提升。例如，可以从经RNAi耗尽特定因子后的细胞中制备提取物，从而深入了解该因子在特定过程中的作用。本章的目标有三个。首先，我们将让读者熟悉高质量、可靠的HeLa细胞细胞质提取物的制备过程。其次，详细描述一种独立提取物标准化的方法，以便进行可靠的提取物间比较。最后，介绍细胞质提取物在检测mRNA周转多个方面的用途和应用。这些程序共同构成了用于哺乳动物细胞RNA降解机制分析的重要工具。
