Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA; Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
Methods. 2018 Mar 15;137:49-54. doi: 10.1016/j.ymeth.2017.11.012. Epub 2017 Dec 1.
Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as "cores" that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.
应激颗粒是一种动态的、保守的非翻译 RNA-蛋白复合物，在细胞应激时形成，与许多神经退行性疾病中的病理性聚集体有关。哺乳动物应激颗粒包含稳定的结构，称为“核心”，可以通过生化方法进行纯化。本文介绍了一种从应激颗粒核心中分离 RNA 进行 RNA-Seq 分析的逐步指南。我们还描述了一种通过单分子 FISH 验证 RNA-Seq 结果的方法，以及如何量化单分子 FISH 结果。这些方案为描述应激颗粒的 RNA 含量提供了一个起点，并可能有助于在各种生物背景下发现应激颗粒的组装机制和功能。
