Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Summary

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

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 ¹. 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 ². 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 ³. 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 ^4-11. 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 ¹². 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.

Protocol

1. RNA Probe Preparation

Overview: The following section describes the steps required to make Digoxigenin (Dig)-labeled RNA probes suitable for FISH. The first step involves cloning or PCR amplifying a sequence corresponding to the transcribed region of a gene of interest that will be used to generate a sequence-specific probe. This can be achieved by first cloning the gene segment into a plasmid in which the multiple cloning site is flanked by bacteriophage promoter elements (T7, T3 or Sp6), which can then serve as a template for PCR using flanking primers that incorporate the promoter sequences, thus generating a linear PCR....

Results

When performed successfully, this procedure offers a strikingly enhanced level of detail in the spatio-temporal analysis of gene expression and mRNA localization dynamics during early Drosophila embryogenesis. Indeed, as illustrated in Figure 3A for the classical pair-rule gene runt (run), one can use this protocol to observe gene expression events via the detection of nascent transcript foci in groups of expressing nuclei. In addition, as shown in the embryo mosaic in

Discussion

For probe synthesis steps, we typically generate run-off antisense RNA probes by in vitro transcription from full-length Drosophila cDNAs amplified from plasmids found in the Drosophila Gene Collection (DGC), a resource detailed at the following website: http://www.fruitfly.org/DGC/index.html ^14,15. This approach has been used extensively in large scale ISH studies aimed at mapping gene expression and mRNA localizat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
T7, T3 or SP6 RNA Polymerase	Fermentas Life Sciences	EP0101,EP0111,EP0131	Kits contain reaction buffer.
DIG RNA Labeling Mix	Roche Applied Science	11 277 073 910
RNAguard	Amersham Biosciences	27-0816-01
3M sodium acetate
Cold 100% ethanol.
Cold 70% ethanol.
Chlorine bleach solution diluted 1:1 with water.
Heptane
Methanol
proteinase K	Sigma Aldrich Oakville, ON, Canada	Catalog No. P2308
40% formaldehyde solution, freshly prepared
PBS-Tween solution (PBT)		1xPBS, 0.1% Tween-20
Glycine solution		2 mg/ ml glycine in PBT
HRP-conjugated mouse monoclonal anti-DIG	Jackson ImmunoResearch Laboratories Inc	200-032- 156	(1/400 dilution of a 1 mg/ml stock solution in PBTB
HRP-conjugated sheep monoclonal anti-DIG	Roche Applied Science, Laval, QC	1 207 733	1/500 dilution of stock solution in PBTB
Biotin-conjugated mouse monoclonal anti-DIG	Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA	200-062-156	(1/400 dilution of a 1 mg/ml stock solution in PBTB
Streptavidin-HRP conjugate	Molecular Probes, Eugene OR, USA	S991	(1/100 dilution of a 1 μg/ml stock