Work conducted in the L&eacute;cuyer laboratory is supported by funding from the National Sciences and Engineering Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR) and the Fonds de Recherche en Sant&eacute; du Qu&eacute;bec (FRSQ). Fabio Alexis Lefebvre and Ga&euml;l Moquin-Beaudry are supported by NSERC undergraduate research studentships, while Carole Iampietro is supported by the Angelo Pizzagalli postdoctoral fellowship.

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 (T...

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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: <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www.fruitfly.org/DGC/index.html</a> 14,15. This approach has been used extensively in large scale ISH studies aimed at mapping gene expression and mRNA localizat...

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

whole mount rna fluorescent in situ hybridization of drosophila embryos

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.

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 <s...

Watch this Scientific Journal Video about Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos at JoVE.com

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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18.1K visualizações.  Institut de Recherches Cliniques de Montréal (IRCM). Aqui nós descrevemos um fluorescente todo-mount In situ Protocolo de hibridização (FISH) para determinar as propriedades de expressão e localização de RNAs expressas durante a embriogênese na mosca da fruta, Drosophila melanogaster.