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

Arbejdet udføres i Lecuyer laboratorium er støttet af midler fra National Sciences and Engineering Council of Canada (NSERC), den canadiske Institutes of Health Research (CIHR) og Fonds de Recherche en Santé du Québec (FRSQ). Fabio Alexis Lefebvre og Gaël Moquin-Beaudry understøttes af NSERC bachelor forskning stipendier, mens Carole Iampietro støttes af Angelo Pizzagalli postdoc-stipendium.

1. RNA Probe Preparation Oversigt: I det følgende afsnit beskrives de trin, der kræves for at gøre digoxigenin (Dig)-mærkede RNA-prober egnede til FISH. Det første trin involverer kloning eller PCR amplifikation af en sekvens svarende til den transkriberede region i et gen af ​​interesse, som vil blive anvendt til at generere en sekvens-specifik probe. Dette kan opnås ved først at klone gensegment i et plasmid, hvori det multiple kloningssted er flankeret af bakteri...

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For probe syntesetrin vi typisk genererer afløb antisense RNA-prober ved in vitro transkription fra fuld længde Drosophila cDNA&#39;er amplificeret fra plasmider fundet i Drosophila Gene Collection (DGC), en ressource beskrevet på følgende websted: <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www .fruitfly.org / DGC / index.html</a> 14,15. Denne fremgangsmåde har været brugt i udstrakt grad i store ISH undersøgelser med henblik på kortlæg...

<table border="1"><tbody><tr><td> Navn på Reagent </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> T7, T3 eller SP6 RNA-polymerase </td><td> Fermentas Life Sciences </td><td> EP0101, EP0111, EP0131 </td><td> Kits indeholder reaktionsbuffer. </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 natriumacetat </td><td></td><td></td><td></td></tr><tr><td> 100% kold ethanol. </td><td></td><td></td><td></td></tr><tr><td> Kold 70% ethanol. </td><td></td><td></td><td></td></tr><tr><td> Blegemiddelopløsning fortyndet 1:1 med vand. </td><td></td><td></td><td></td></tr><tr><td> Heptan </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> Katalog nr. P2308 </td><td></td></tr><tr><td> 40% formaldehydopløsning, friskfremstillet </td><td></td><td></td><td></td></tr><tr><td> PBS-Tween-opløsning (PBT) </td><td></td><td> 1XPBS, 0,1% Tween-20 </td><td></td></tr><tr><td> Glycinopløsning </td><td></td><td> 2 mg / ml glycin i PBT </td><td></td></tr><tr><td> HRP-konjugeret muse-monoklonalt anti-DIG </td><td> Jackson ImmunoResearch Laboratories Inc </td><td> 200-032 - 156 </td><td> (1/400 fortynding af en 1 mg / ml stamopløsning i PBTB </td></tr><tr><td> HRP-konjugeret fåre monoklonalt anti-DIG </td><td> Roche Applied Science, Laval, QC </td><td> 1 207 733 </td><td> 1/500 fortynding af stamopløsning i PBTB </td></tr><tr><td> Biotinkonjugeret muse-monoklonalt anti-DIG </td><td> Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA </td><td> 200-062-156 </td><td> (1/400 fortynding af en 1 mg / ml stamopløsning i PBTB </td></tr><tr><td> Streptavidin-HRP-konjugat </td><td> Molecular Probes, Eugene OR, USA </td><td> S991 </td><td> (1/100 fortynding af en 1 mg / ml stock </td></tr></tbody></table>

whole mount rna fluorescent in situ hybridization of drosophila embryos

Vurdering af ekspressionsmønstret af et gen, såvel som de subcellulære lokalisering egenskaber af sine transskriberede RNA, er vigtige funktioner til at forstå dens biologiske funktion under udvikling. RNA in situ hybridisering (RNA-ISH) er en kraftfuld metode, der anvendes til at visualisere RNA fordeling egenskaber, det være sig på de organismal, cellulære eller subcellulære niveauer 1. RNA-ISH er baseret på hybridiseringen af en mærket nukleinsyreprobe (fx antisense-RNA, oligonucleotider) komplementær til sekvensen af et mRNA eller et ikke-kodende RNA-mål af interesse 2. Da proceduren kræver primære sekvensinformation alene at generere sekvensspecifikke prober, kan det generelt anvendes i en bred vifte af organismer og vævsprøver 3. Faktisk har en række store ISH undersøgelser blevet gennemført for at dokumentere genekspression og RNA lokalisering dynamik i forskellige modelorganismer, hvilket har ført tiletablering af vigtige samfunds ressourcer 4-11. Mens en række af sonde mærkning og detektion strategier er blevet udviklet gennem årene, den kombinerede anvendelse af fluorescens-mærkede påvisningsreagenser og enzymatiske signalforstærkning trin giver betydelige forbedringer i følsomhed og opløsning af proceduren 12. Her beskriver vi en optimeret fluorescerende in situ-hybridisering fremgangsmåde (FISH) under anvendelse af tyramid signalforstærkning (TSA) for at visualisere RNA-ekspression og lokalisering dynamikken i iscenesatte Drosophila-embryoner. Fremgangsmåden udføres i 96-brønds PCR-plade-format, hvilket i høj grad letter samtidig behandling af et stort antal prøver.

Hvornår gennemført, denne procedure giver en markant forbedret niveau af detaljer i den spatio-temporale analyse af genekspression og mRNA lokalisering dynamik i den tidlige Drosophila embryogenese. Som vist i figur 3A for den klassiske pair-reglen genet runt (run), kan man anvende denne protokol til at observere genekspression begivenheder via påvisning af nascent transcript foci i grupper af ekspression kerner. Desuden, som vist i embryonet mosaik i figur 3B fremg...

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

Her beskriver vi en hel-mount fluorescerende<em&gt; In situ</em&gt; Hybridisering (FISH)-protokollen til bestemmelse af udtrykket og lokalisering egenskaber af RNA&#39;er udtrykt under embryogenese hos bananfluen,<em&gt; Drosophila melanogaster</em&gt;.

Hele Mount RNA Fluorescent<em&gt; In situ</em&gt; Hybridisering af<em&gt; Drosophila</em&gt; Embryoner

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

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

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

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

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

Påvisning af viralt RNA ved fluorescens<em&gt; In situ</em&gt; Hybridisering (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 af enkeltcelle-gentransskription ved RNA Fluorescent<em&gt; In Situ</em&gt; Hybridisering (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

Kromosom Replikerer Timing Kombineret med Fluorescent<em&gt; In situ</em&gt; Hybridisering

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; Hybridisering i Hele Mount Embryoner og Cell Histologi Tilpasset til Marine bruskfisk

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

High Resolution Hele Mount<em&gt; In Situ</em&gt; Hybridisering inden Zebrafisk embryoner at studere genekspression og funktion

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

Analyse af apoptose i zebrafisk embryoner Whole-mount Immunofluorescens Detect Aktiveret Caspase 3

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