Whole Mount RNA Fluorescent <em>in situ</em> Hybridization of <em>Drosophila</em> Embryos

Abstract

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

“…Embryos at 0 to 2 h from wild-type fly y, w, hrp38 mutant (Hrp38d05717/Df), and Parg Ϫ/Ϫ germ line clones generated through the FLP-DFS method were collected for nos RNA fluorescence in situ hybridization (FISH) using a published protocol (33). DIG-labeled nos antisense probe was produced with a DIG RNA labeling kit (SP6/T7; Roche).…”

RNA transcribed from heterochromatic simple-tandem repeats are required for male fertility and histone-protamine exchange in Drosophila melanogaster

“…RNA-FISH probe hybridization and primary antibody incubation. RNA probe hybridization for all tissues was carried about according to 27…”

“…For RNA-FISH of repeat RNAs, 0-8hr Oregon R embryos were collected on apple juice plates, dechorionated and processed according to 27 , as per protocols 1 and 3 above, with the exception of using 37% formaldehyde stock from Sigma (cat. F1635-500ML).…”

Expression of phosphatase of regenerating liver family genes during embryogenesis: an evolutionary developmental analysis among Drosophila, amphioxus, and zebrafish

“…Template preparation, probe synthesis, and procedure for whole mount in situ hybridization were performed as previously described [30]. All embryos were observed under a Nikon Eclipse E800 microscope equipped with Nomarski differential interference contrast optics and a CCD camera.…”