Lin Pei-Hsun, Singh Dharam, Bernstein Jonathan A, Lin-Chao Sue
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.
Methods Enzymol. 2008;447:47-64. doi: 10.1016/S0076-6879(08)02203-9.
The decay of mRNA plays an important role in the regulation of gene expression. Although relatively ignored for many years and regarded as a simple ribonucleotide salvage pathway, mRNA decay has been established in recent years as a well-defined cellular process that plays an integral role in determining gene expression. The recent application of microarray methods to the study of diverse organisms will help us to better understand these gene regulatory circuits and the influence of transcript stability on gene expression. DNA microarray technology is the method of choice to study individual mRNA half-lives on a global scale. It is important to standardize these methods to generate reproducible and reliable results. In this chapter, we describe experimental designs for the analysis of mRNA decay on a genome-wide scale and provide detailed protocols for each experimental step. We also present an analysis of the decay of chromosomally encoded mRNAs in E. coli.
mRNA的降解在基因表达调控中起着重要作用。尽管多年来相对被忽视且被视为一种简单的核糖核苷酸补救途径,但近年来mRNA降解已被确立为一个明确的细胞过程,在决定基因表达中发挥着不可或缺的作用。微阵列方法最近在多种生物体研究中的应用将有助于我们更好地理解这些基因调控回路以及转录本稳定性对基因表达的影响。DNA微阵列技术是在全球范围内研究单个mRNA半衰期的首选方法。对这些方法进行标准化以产生可重复和可靠的结果很重要。在本章中,我们描述了用于全基因组范围内mRNA降解分析的实验设计,并为每个实验步骤提供了详细的方案。我们还对大肠杆菌中染色体编码mRNA的降解进行了分析。