Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France.
Methods Mol Biol. 2021;2300:73-85. doi: 10.1007/978-1-0716-1386-3_8.
The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.
确定非编码 RNA（ncRNA）的细胞定位对于解析其功能非常有帮助。RNA-FISH 是一种在固定细胞中检测特定 RNA 的强大方法。它可以在单个细胞和组织中定位和定量 RNA 分子。还开发了更精细的 RNA-FISH 方法来确定 RNA 的转录和降解速率。本章描述了我们开发的一种 RNA-FISH 方案，用于研究卫星 III（SATIII）RNA 的表达和定位。这种特定类型的 ncRNA 是在响应各种细胞应激，包括热休克时表达的。该方案基于使用生物素化的锁核酸（LNA）探针，随后通过链霉亲和素，Alexa Fluor 488 缀合物进行检测。还描述了一种允许通过免疫荧光术有效结合 RNA-FISH 和蛋白质检测的方案，以及我们最近开发的用于自动荧光信号分析的生物信息学分析流程 Substructure Analyzer。
