Name: detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture
Uploaded: 2016-07-22T15:00:00
Description: Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Dette arbeidet ble finansiert av Singapore NRF fellesskap (NRF-2011NRF-NRFF001-025) samt CBRG (NMRC / CBRG / 0070/2014).

MERK: RNA av interesse i forbindelse med denne studien er et fragment av AR 3&#39;UTR. 1. Fremstilling av biotin-merket RNA <ol><li> For å få RNA, forsterke T7-AR-oligo med PCR, etterfulgt av in vitro transkripsjon ved å bruke et kommersielt kit og følge produsentens instruksjoner 11. MERK: Grunning for PCR er oppført i Tabell 4: T7-AR-F og T7-AR-R. </li><li> For ikke-spesifikk binding kontroll, forsterke en partiell sekvens av ildfluelucife...

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lncRNAs og RBPs har viktige roller i helse og sykdom, men de molekylære mekanismene for disse molekylene er dårlig forstått. Identifisering av proteiner som samhandler med lncRNA molekyler er et viktig skritt mot å belyse de reguleringsmekanismer. Anrikning av RBPs av RNA pull-down analysesystem er basert på in-oppløsning fangst til å samvirke komplekset slik at proteiner fra et cellelysat selektivt kan ekstraheres ved bruk av biotinylerte RNA agn som er bundet til streptavidin magnetiske kuler. Flere andre metod...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RBPs er proteiner som binder seg til det dobbelte eller enkelt-trådet RNA i celler og deltar i dannelse av RNA-proteinkomplekser. RBPs kan binde en rekke RNA arter, inkludert mRNA og lange ikke-kodende RNA (lncRNAs), og utøve sin innflytelse på post-transkripsjonsnivåer. Begge RBPs og lncRNAs er fremstår som nye regulatorer i adipose utvikling og funksjon 1,2,3. For å forstå mekanismen for RBP- og lncRNA-medierte regulering av cellulære veier, det er ofte nødvendig for å påvise interaksjon mellom et spesifikt RNA-molekyl og ett eller flere RBPs, og, noen ganger, for å identifisere hele spekteret av proteinpartnere for en RNA transkripsjon. Imidlertid kan eksperimentet være utfordrende på grunn av det høye innhold av lipider i adipocytter. RNA pull-down protokollen som er beskrevet her kan anvendes for å hente proteinpartnere for en spesifikk RNA fra fettceller lysatene av primære kulturer 4,5. Begrunnelsen for dette protocol er oppsummert som følger. Et RNA agn er in vitro transkribert fra et DNA-templat, biotin-merket og konjugert til streptavidin-belagte magnetiske kuler 4. RNA agn inkuberes med cellelysatene for å muliggjøre dannelse av RNA-proteinkomplekser, som deretter trekkes ned på en magnetisk stativ. Mer spesielt beskrives det RNA-pull-down-protokollen nedenfor bruke et RNA-fragment fra AR 3&#39;UTR som agn for å hente Hur protein, et universelt uttrykt RBP bundet til 3&#39;UTR av mRNA 6,7, fra theadipocytelysate av primær kultur. For å teste bindende spesifisitet av denne analysen, er en ikke-relevante RNAas samt tomme streptavidin perler inkludert som kontroll. Denne protokollen er kompatibel med western blotting eller massespektrometri (MS) for å bekrefte fangst av en spesifikk RBP eller for å identifisere den fullstendige repertoar av fangede RBPs, henholdsvis 8. Flere teknikker er tilgjengelige for å studere RNA-proteininteraksjons. For å avdekke RNA er bundet av en gitt RBP, RIP (RNA immunoprecipitation) og CLIP (UV kryssbinding og immunoprecipitation) kan brukes. I motsetning til å identifisere protein partnere i et gitt RNA, RNA pull-down, kvitre (kromatin isolering av RNA rensing), SKJEMA (fangst hybridisering analyse av RNA mål) og RAP (RNA anti rensing) kan brukes. Sammenlignet med de senere meldinger, tar RNA rullegardin teknikk mindre innsats for å sette opp. Den kan benyttes for å fange proteiner in vitro og in vivo. In vivo tilnærming av RNA pull-down, som er teknisk mer krevende enn in vitro motstykke, bevarer RNA-protein interaksjoner ved kryssbinding i celler, fanger aptamer-merket RNA av interesse fra cellene, og senere oppdager innbundne RBPs. RNA pull-down kan anvendes for å berike lave rikelig RBPs, og for å isolere og identifisere RNA-proteinkomplekser som har forskjellige funksjonelle roller i å kontrollere celleregulering 9,10 </sopp&gt;.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Major materials used in this study.</td>
			<td style="width:177px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript kit</td>
			<td>&#160;Ambion</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Biotin-14-CTP&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucAway spin columns&#160;</td>
			<td>Ambion</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HuR (3A2) mouse monoclonal antibody (sc-5261)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG-HRP(sc-2005)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypure Molecular Biology Grade Water (nuclease-free)&#160;</td>
			<td>HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (1&#215;PBS)</td>
			<td>&#160;HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase inhibitor&#160;</td>
			<td>Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pfu Turbo DNA polymerase&#160;</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIsure</td>
			<td>&#160;Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Major equipment used in this study.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend Micro 21R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend X1R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Rad T100 thermal cycler&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BOECO rotator Multi Bio RS-24&#160;</td>
			<td>BOECO,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein gel electrophoresis and blotting apparatus&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 magnet&#160;</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA-bindende proteiner (RBPs) er fremstår som en regulerende lag i utvikling og funksjon av adipose. RBPs spille en nøkkelrolle i genuttrykk regulering på posttranskripsjonelt nivåer ved å påvirke stabiliteten og translasjonsforskning effektiviteten av målet mRNA. RNA pull-down teknikken har blitt mye brukt for å studere RNA og protein, noe som er nødvendig for å belyse mekanismen bak RBPs &#39;samt lang ikke-kodende RNA&#39; (lncRNAs) funksjon. Men den høye lipid overflod i adipocytter utgjør en teknisk utfordring i å gjennomføre dette eksperimentet. Her en detaljert RNA pull-down-protokollen er optimalisert for primær adipocyte kultur. En RNA-fragment fra androgen reseptor-tallet (AR) 3 &#39;ikke-translatert område (3&#39;UTR) som inneholder en adenylate-uridylate rike elementwas brukt som et eksempel for å demonstrere hvordan du henter sin RBP partner, Hur protein, fra adipocyte lystate. Den fremgangsmåte som er beskrevet her kan anvendes for å påvise interaksjonen mellomRBPs og ikke-kodende RNA, samt mellom RBPs og koding RNA.

I denne demoen eksperiment ble en AR RNA fragment brukt som agn for å fange sin bindende protein Hur. Både en FL RNA agn som er ikke-relevant for Hur protein, og en porsjon av konjugerte tomme streptavidin perler tjente som negative kontroller. RNA-protein interaksjoner kan skje enten i kjernen eller i cytoplasma, og denne rullegardin protokoll kan anvendes enten samlet eller fraksjonerte (nukleær eller cytoplasmatiske) cellelysater. Analyse av proteiner ved western blotting viser at ...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

En RNA pull-down-protokollen er optimalisert her for påvisning av vekselvirkninger mellom RNA-bindende proteiner (RBPs) og ikke-kodende samt koding RNA. Et RNA-fragment fra androgenreseptoren (AR) ble anvendt som et eksempel for å vise hvordan man skal hente sine RBP fra lystate av primære brune adipocytter.

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RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

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