Name: quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation
Uploaded: 2014-11-26T14:00:00
Description: Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Probe Forberedelse <ol><li> Opnå flere unikke oligonukleotider i Xist (20-30 nukleotid-længde oligonukleotider, 63-65 ºC smeltende temperatur, tabel 1) med en 5&#39;-amino modifikation og suspendere i vand. </li><li> Pool ækvimolære mængder af oligonukleotider med 5&#39;-amino modifikation (total 4,5 ug) og mærke med amin-reaktivt fluorescerende farvestof efter fabrikantens anvisninger. <ol><li> Opløses de poolede oligonukleotider i sidste 5 pi nuclease-frit vand, og der tilsættes 3 pi 1 M...

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I dette papir har vi præsenteret en hurtig immuno-FISH protokol, der tager mindre end 5 timer at fuldføre præparatglaspræpareringsprotokol, Xist RNA FISH, og immunofluorescens for H3K27me3. I forhold til den generelle immuno-FISH metode til Xist RNA påvisning, hvilket skal normalt inkubation natten for RNA FISH, denne protokol ikke blot reducerer tidsforbrug, men også forbedrer følsomheden af ​​immun-FISH anvendelse af oligonukleotidprober. Da Xist er stærkt transkriberet under X...

Mammalian X-kromosom inaktivering (XCI) er en strategi til at kompensere for ubalancen i X-bundet gen dosering mellem XX og XY, hvor én af de to X-kromosomer hos kvinder er transkriptionelt inaktiveret 1. X-kromosom inaktivering er en stor model for lang ikke-kodende RNA (lncRNA) forskning. X-kromosom inaktivering reguleres af flere lncRNAs, og er blevet grundigt undersøgt i de seneste årtier for at afdække de crosstalk mekanismer mellem lncRNAs, transskription, kromatin struktur og nukleart organisation 2, 3. X inaktivering center (XIC) placeret på X-kromosomet er en kompleks genetisk locus består af en række gener, der producerer ikke-kodende RNA&#39;er 4. X-inaktiv specifik udskrift (Xist) lncRNA i eutherian pattedyr er en sådan lncRNA som spiller en afgørende rolle i X-kromosom inaktivering 5,6. Xist transkripter omgiver placeringen af ​​futuren Xi at indlede X-kromosom inaktivering, og fremstår som en sky, når visualiseret ved hjælp af RNA FISH; denne formation benævnt &quot;Xist Cloud&quot; 7. Da Xist RNA interagerer med forskellige kromatin-modificerende enzymer, observeres co-lokalisering af Xist skyer med forskellige epigenetiske modifikationer til lydløs kromatin og undertrykkende transskription under X-kromosom inaktivering 8. For eksempel Xist RNA interagerer med Polycomb undertrykkende kompleks 2 (PRC2), som er ansvarlig for H3K27me3 og inducerer en undertrykkende kromatin tilstand 9. Forekomsten af Xist sky på Xi og dets co-lokalisering med den intensive H3K27me3 modifikation udgør en fakultativ heterochromatin landskab af Xi 10,11. Cytogenetiske teknikker, såsom DNA / RNA FISH er kommet en lang vej fra den traditionelle metode ved hjælp af radioaktivt mærkede prober 12 de seneste og avancerede teknikker med forbedret følsomhed ogfluorescerende billeddannelse ved hjælp af flere oligonukleotidprober 13,14. DNA / RNA-FISH kombineret med immunfluorescens er rutinemæssigt blevet anvendt som et cytologisk redskab til at forstå spatiotemporale nukleare organisation, RNA lokalisering, chromatin struktur og modifikationer. Den mest standard forberedelse probe for RNA FISH indebærer anvendelse af plasmid eller bakterielle kunstige kromosom (BAC) kloner og deres efterfølgende mærkning med enten nick translation eller tilfældig priming 15. Imidlertid næsten 70% af gener i mus og 40% af gener i mennesker viser en overlapning af sense- og antisense transkripter 16 og dermed kræver en streng-specifik FISH metode for at skelne sense- og antisense transkripter. In vitro transskriberede RNA-prober (riboprobe ) bruges ofte til streng-specifikke RNA FISH 17,18; men dette indebærer fremstilling af en plasmidklon eller PCR-produkt med T7, SP6, eller T3-promotor og syntetiserer riboprober. Endvidere riboprober afledt fra genomic DNA eller cDNA indeholder ofte ikke-specifikke regioner og repetitive elementer, der resulterer i høj baggrundsstøj. Et andet problem er, at riboprober, som er et par hundrede nukleotider i længden og indeholder flere fluoroforer, ikke effektivt kan trænge ind i kernen. For at omgå dette har flere kortere oligonukleotidprober mærket med en enkelt fluorophor i slutningen blevet udviklet, som har en god følsomhed, ensartet signalstyrke og let rensning og håndtering 14. Derudover er DNA-oligonukleotider er generelt mere stabile end RNA. Vi har anvendt en lignende strategi for at anvende oligonucleotider i Xist RNA FISH 19 med immunofluorescens at forstå epigenetiske dynamik Xi induceret af Xist lncRNA under processen med X-kromosom inaktivering. Denne protokol beskriver skabelsen af ​​oligonukleotidprober og ordentlig forberedelse af celler, samt udnyttelse af immunofluorescens og RNA fisk. Xist RNA FISH Brug af flere oligonucleotides er omkostningseffektiv tilgang på lang sigt, hvis Xist RNA FISH udføres rutinemæssigt i ens laboratorium. Denne teknik kan anvendes til at identificere lncRNAs i celler, samtidig med at kortlægge dens co-lokalisering med epigenetiske ændringer eller faktorer. En væsentlig fordel ved protokollen er mulighed for nemt at ændre det, der passer til ens forskningsinteresser.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

Ved at kombinere RNA fluorescerende in situ-hybridisering (FISH) med immunofluorescens (immuno-FISH) skaber en teknik, der kan være ansat på enkelt celle niveau til sporing af rumlig dynamik RNA lokalisering med samtidig indsigt i lokalisering af proteiner, epigenetiske modifikationer og andre detaljer der kan fremhæves ved immunfluorescens. X-kromosom inaktivering er et paradigme for lang ikke-kodende RNA (lncRNA) -medieret gendæmpning. X-inaktiv specifik udskrift (Xist) lncRNA akkumulering (kaldet en Xist sky) på en af ​​de to X-kromosomer i hunner pattedyr er et afgørende skridt til at indlede X-kromosom inaktivering. Xist RNA direkte eller indirekte interagerer med forskellige kromatin-modificerende enzymer og indfører forskellige epigenetiske landskaber til det inaktive X-kromosom (Xi). En kendt epigenetisk kendetegnende for Xi er histon H3 trimethyl-lysin 27 (H3K27me3) ændring. Her beskriver vi en enkel og hurtig immuno-FISHProtokol til påvisning Xist RNA ved hjælp af RNA FISH med flere oligonucleotidsonder kombineret med immunfluorescens af H3K27me3 at undersøge lokalisering af Xist RNA og tilknyttede epigenetiske modifikationer. Anvendelse af oligonukleotidprober resulterer i en kortere inkubationstid og mere følsom detektion af Xist RNA sammenlignet med in vitro transskriberede RNA-prober (riboprober). Denne protokol giver et stærkt redskab til at forstå dynamikken i lncRNAs og de tilhørende epigenetisk modifikation, chromatinstruktur, nuklear organisation og transkriptionel regulering.

Repræsentative billeder af hurtig immuno-fisk er vist i figur 1A. Co-lokaliseringen af ​​Xist RNA sky og H3K27me3 signal på Xi blev påvist i differentierende kvindelige celler. På dag 12 efter differentiering, mere end 90% af EB-celler havde en Xist cloud (figur 1B). Short oligonukleotidprober effektivt trængt ind i kerner, hvilket fører til en visualisering af næsten alle H3K27me3 signaler co-lokaliseres med Xist RNA (figur 1C, 97%, n = 150). <p class="j...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Quick Fluorescent<em&gt; In Situ</em&gt; Hybridisering Protokol for Xist RNA Kombineret med Immunofluorescens af histon Ændring i X-kromosom Inaktivering

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

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Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

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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 ...