Name: whole mount rna fluorescent in situ hybridization of drosophila embryos
Uploaded: 2013-01-30T13:00:00
Description: Watch this Scientific Journal Video about Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos at JoVE.com

Werkzaamheden die in het Lecuyer laboratorium wordt ondersteund door financiering van de National Sciences and Engineering Council of Canada (NSERC), de Canadese Institutes of Health Research (CIHR) en het Fonds de Recherche en Sante du Quebec (FRSQ). Fabio Alexis Lefebvre en Gaël Moquin-Beaudry worden ondersteund door NSERC undergraduate research studiebeurzen, terwijl Carole Iampietro wordt ondersteund door de Angelo Pizzagalli postdoctoraal fellowship.

1. Bereiding RNA Probe Overzicht: De volgende sectie beschrijft de stappen die nodig zijn om Digoxigenine (Dig)-gemerkte RNA-probes die geschikt zijn voor FISH te maken. De eerste stap betreft het kloneren of PCR amplificatie van een sequentie die overeenkomt met het getranscribeerde gebied van een gen van interesse dat wordt gebruikt om een ​​sequentie-specifieke probe te genereren. Dit kan worden bereikt door eerst het kloneren van het gen segment in een plasmide waarin ...

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Voor sonde synthese stappen, hebben we meestal het genereren van run-off antisense RNA probes door in vitro transcriptie van volledige lengte Drosophila cDNA geamplificeerd van plasmiden in de Drosophila Gen (DGC), een bron beschreven op de volgende website: <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www .fruitfly.org / DGC / index.html</a> 14,15. Deze benadering is veelvuldig gebruikt in grootschalige ISH studies gericht op het in kaart brengen van gene...

<table border="1"><tbody><tr><td> Naam van de Reagens </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Opmerkingen (optioneel) </td></tr><tr><td> T7, T3 of SP6 RNA polymerase </td><td> Fermentas Life Sciences </td><td> EP0101, EP0111, EP0131 </td><td> Kits bevatten reactie 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 natriumacetaat </td><td></td><td></td><td></td></tr><tr><td> Koude 100% ethanol. </td><td></td><td></td><td></td></tr><tr><td> Koude 70% ethanol. </td><td></td><td></td><td></td></tr><tr><td> Chloor oplossing 1:1 met water verdund. </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 oplossing, vers bereid </td><td></td><td></td><td></td></tr><tr><td> PBS-Tween oplossing (PBT) </td><td></td><td> 1xPBS, 0,1% Tween-20 </td><td></td></tr><tr><td> Glycine oplossing </td><td></td><td> 2 mg / ml glycine in PBT </td><td></td></tr><tr><td> HRP-geconjugeerd muis monoklonaal anti-DIG </td><td> Jackson ImmunoResearch Laboratories Inc </td><td> 200 tot 032 - 156 </td><td> (1/400 verdunning van een 1 mg / ml voorraadoplossing in PBTB </td></tr><tr><td> HRP-geconjugeerd schapen monoklonale anti-DIG </td><td> Roche Applied Science, Laval, QC </td><td> 1 207 733 </td><td> 1/500 verdunning van de voorraadoplossing in PBTB </td></tr><tr><td> Biotine-geconjugeerd muis monoklonaal anti-DIG </td><td> Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA </td><td> 200-062-156 </td><td> (1/400 verdunning van een 1 mg / ml voorraadoplossing in PBTB </td></tr><tr><td> Streptavidine-HRP conjugaat </td><td> Molecular Probes, Eugene OR, USA </td><td> S991 </td><td> (1/100 verdunning van een 1 ug / ml stock </td></tr></tbody></table>

whole mount rna fluorescent in situ hybridization of drosophila embryos

Beoordelen het expressiepatroon van een gen, alsmede de subcellulaire lokalisatie eigenschappen van het getranscribeerde RNA, zijn kenmerkend voor het begrijpen van de biologische functie tijdens de ontwikkeling. RNA in situ hybridisatie (ISH-RNA) is een krachtige methode voor het visualiseren RNA distributie eigenschappen, ten organismale cellulaire of subcellulaire niveau 1. RNA-ISH is gebaseerd op de hybridisatie van een gemerkte probe nucleïnezuur (bijvoorbeeld antisense RNA, oligonucleotiden) complementair aan de sequentie van een mRNA of een niet-coderende RNA doelwit van belang 2. De procedure vereist primaire sequentie informatie alleen te genereren sequentie-specifieke probes, kan algemeen worden toegepast op een groot aantal organismen en weefselmonsters 3. Uit een aantal grootschalige ISH studies zijn uitgevoerd om genexpressie documenteren en lokalisatie dynamiek RNA in verschillende modelorganismen, heeft dat tot devestiging van belangrijke communautaire middelen 4-11. Hoewel verschillende probe labeling en detectie strategieën zijn ontwikkeld de afgelopen jaren de gecombineerde gebruik van fluorescentie-gemerkt detectiereagentia en enzymatische signaalversterking stappen bieden significante verbeteringen in de gevoeligheid en resolutie van de procedure 12. Hier beschrijven we een geoptimaliseerd fluorescente in situ hybridisatie methode (FISH) gebruik tyramide signaalamplificatie (TSA) om RNA expressie en lokalisatie dynamiek opgevoerd Drosophila embryo zichtbaar. De procedure wordt uitgevoerd in 96-well plaat formaat PCR, die aanzienlijk vergemakkelijkt het gelijktijdig verwerken van grote aantallen monsters.

Wanneer deze wordt uitgevoerd met succes, deze procedure biedt een opvallend hoger niveau van detail in de spatio-temporele analyse van genexpressie en mRNA lokalisatie dynamiek tijdens de vroege embryogenese van Drosophila. Zoals geïllustreerd in figuur 3A van de klassieke pair-rule gen runt (run), kan men dit protocol om genexpressie gebeurtenissen observeren via de detectie van ontluikende transcript foci in groepen expressie kernen. Bovendien, zoals getoond in de embryo mozaïek i...

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

Hier beschrijven we een geheel gemonteerde fluorescent<em&gt; In situ</em&gt; Hybridisatie (FISH)-protocol voor het bepalen van de expressie en lokalisatie eigenschappen van RNA&#39;s die tijdens de embryogenese in de fruitvlieg,<em&gt; Drosophila melanogaster</em&gt;.

Hele Mount RNA TL<em&gt; In situ</em&gt; Hybridisatie van<em&gt; Drosophila</em&gt; Embryo&#39;s

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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Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Double Whole Mount in situ Hybridization of Early Chick Embryos

Dubbele Hele Mount in situ hybridisatie van het vroege embryo&#39;s Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebravis hele Mount High-Resolution Double Fluorescent In Situ Hybridisatie

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Met behulp van Whole Mount In situ Hybridisatie aan Moleculaire Biologie en organismale Link

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Hele Mount<em&gt; In situ</em&gt; Hybridisatie van E8.5 aan E11.5 muis embryo&#39;s

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

Detectie van virale RNA door fluorescentie<em&gt; In situ</em&gt; Hybridisatie (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Analyse van de Single-cell gentranscriptie door RNA TL<em&gt; In Situ</em&gt; Hybridisatie (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosoom repliceren Timing In combinatie met TL-<em&gt; In situ</em&gt; Hybridisatie

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

RNA<em&gt; In situ</em&gt; Hybridisatie in Hele Mount Embryo&#39;s en histologie Aangepast voor Marine kraakbeenvissen

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Hoge resolutie Hele Mount<em&gt; In Situ</em&gt; Hybridisatie in zebravis embryo&#39;s om genexpressie en functie te bestuderen

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...