Lécuyer Eric
Institut de Recherches Cliniques de Montréal (IRCM), Ouest Montréal, QC, Canada.
Methods Mol Biol. 2011;714:31-47. doi: 10.1007/978-1-61779-005-8_3.
Tissue-specific gene expression is a major determinant in the elaboration of cells with distinctive phenotypes and functions, which is crucial for the development and homeostasis of multicellular organisms. Fluorescent in situ hybridization (FISH) is a powerful method for assessing the expression and localization properties of RNA at subcellular resolution in whole mount organism and tissue specimens. This chapter describes a high-resolution FISH protocol for the detection of RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster. The approach utilizes tyramide signal amplification (TSA) for enhanced sensitivity and resolution in the detection of coding and noncoding RNAs, for the codetection of different RNA species or of RNA and a protein marker of interest. Furthermore, the protocol outlines details for conducting FISH in microtiter plates, which greatly enhances the throughput, practicality, and economy of the procedure.
组织特异性基因表达是形成具有独特表型和功能的细胞的主要决定因素，这对于多细胞生物的发育和体内平衡至关重要。荧光原位杂交（FISH）是一种强大的方法，可在整装生物体和组织标本中以亚细胞分辨率评估RNA的表达和定位特性。本章介绍了一种高分辨率FISH方案，用于检测果蝇胚胎和组织中RNA的表达和定位动态。该方法利用酪胺信号放大（TSA）来提高检测编码和非编码RNA、共检测不同RNA种类或RNA与感兴趣的蛋白质标记物时的灵敏度和分辨率。此外，该方案概述了在微孔板中进行FISH的详细步骤，这大大提高了该过程的通量、实用性和经济性。
