Subcellular transcript localization in Drosophila embryos and tissues visualized by multiplex-fish

Abstract

Determining the spatiotemporal expression dynamics of a gene, or the subcellular localization properties of its encoded RNA, is often a key first step toward elucidating its function. Fluorescent in situ hybridization (FISH) represents the gold standard method for visualizing RNA expression and subcellular localization features in distinct cells, tissue specimens, and whole-mount organisms. This chapter describes a high-resolution FISH protocol for the detection of coding or noncoding RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster . Variations of the protocol are proposed for the co-detection of different RNAs and for the co-labeling of RNAs and proteins. The protocol also outlines details for conducting FISH in 96-well plate format, which significantly enhances the throughput and versatility of the procedure.