Name: rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling
Uploaded: 2015-04-11T14:00:00
Description: Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

This work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC: 418391-2012). AC receives a scholarship from the Centre th&#233;matique de recherche en Neurosciences (CTRN). HDB is funded by a scholarship from the Fonds de Recherche en Sant&#233; du Qu&#233;bec (FRSQ) and ML is a FRSQ Chercheur-Boursier.

BEMÆRK: Forsøgene blev udført i overensstemmelse med den canadiske Guide til pleje og anvendelse af forsøgsdyr og blev godkendt af Udvalget for Université Laval Animal Protection. 1. Prøvefremstilling BEMÆRK: I dette eksempel brugte vi hjerner musen på postnatal dag 1. <ol><li> Brug hypotermi anæstesi i knust is i 3 til 4 min for hvalpe, så dissekere hjerner så hurtigt som muligt i iskold L15 medium. Udfør dette trin inden for 2 min. </li><li> Overf...

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Det mest kritiske punkt af denne metode består i mærkning celler af interesse samtidig forhindre RNA-nedbrydning. Da RNAser er aktive i vandige opløsninger, faldende inkubationstider forbedrer RNA bevaring 11,15. Alle reagenser, der skal behandles med DEPC at inaktivere RNAser. Alle materialer og overflader skal behandles med en overflade RNAse dekontaminering løsning for at forhindre RNAser kontaminering. Endelig, under disse betingelser, tilsætning af RNAse inhibitor i alle opløsninger er effektiv i a...

Hjernen består af en lang række forskellige neuron typer danner komplekse netværk. Disse neuroner er organiseret i forskellige grupper og undergrupper efter deres morfologi, tilslutningsmuligheder og genekspression mønster 1. Udviklingen af microarrays, næste generation sekventering, og QRT-PCR tilbydes mulighed for at sammenligne genekspression profiler af neuronale populationer i forskellige biologiske sammenhænge 1,2. Disse følsomme analyser kræver præcis identifikation og isolering af celletyper af interesse samtidig holde RNA integritet 2-4. Fluorescerende aktiveret cellesortering (FACS) teknik er i vid udstrækning blevet anvendt til at isolere specifikke celletyper baseret på celleoverflademarkører og / eller morfologi. FACS kræver en celle dissociation trin forud for sortering, hvilket resulterer i et fuldstændigt tab af rumlig opløsning 5. Mange neuronale subpopulationer skelnes fra hinanden efter deres anatomiske fordeling i the hjernen. Laser capture mikrodissektion anvendt på tynde hjernesnit giver en passende mulighed for specifik isolering af celler med høj rumlig opløsning 6-8. En væsentlig begrænsning af LCM har været behovet for at identificere celler af interesse baseret på morfologiske kriterier eller fluorescerende protein reportere gensplejsede i dyremodeller. Udviklingen af ​​nye teknikker til at udføre hurtige immunolabeling metoder, bevarede RNA integritet, kombineret med LCM, tillader nu isolering af cellesubpopulationer at gå videre med gen-profilering eksperimenter. Denne fremgangsmåde er tidligere blevet udført, og kort beskrevet i nogle publikationer 1,9-13. Her viser vi en detaljeret procedure for at opnå høj kvalitet RNA fra en specifik undergruppe af celler i en kompleks vævsstruktur ved at kombinere hurtig immunmærkning med LCM. Vi viser, hvordan du udfører vigtige kritiske skridt til at opnå maksimal RNA genvinding og undgå RNA nedbrydning, da det kan SIGligt effekt genekspression profilering. Til demonstration af denne protokol blev dopaminerge neuroner fra en dage gammel mus midthjernen målrettet. Dopaminerge neuroner kan immunolabeled anvendelse af et antistof rettet mod tyrosin hydroxylase (TH), det hastighedsbegrænsende enzym for dopamin-syntese. Efter TH immunfarvning, kan enkelte eller grupper af dopaminerge neuroner derefter isoleres ved hjælp af LCM. Mikrodissekterede celler opsamles i lysepuffer, og RNA ekstraheres ved hjælp af en RNA isolation kit. Kvaliteten og mængden af ekstraheret RNA måles ved hjælp af en Bioanalyzer 5. Yderligere analyse af genekspression ved anvendelse: RNA-sekventering, microarray, eller QRT-PCR kan efterfølgende udføres 2,4,6. Som et eksempel, er en to-trins QRT-PCR demonstreret på laser fanget isoleret dopaminerge domæner. Relativ kvantificering af ekspressionsniveauer af to dopaminerge neuron markørgener illustrerer selektivitet af denne protokol.

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			<td height="42" style="height:42px;width:228px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td style="width:420px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Membrane coated slides&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">11505158</td>
			<td>MembraneSlides PEN-Membrane 2,0 &#181;m; 50 pcs</td>
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			<td height="42" style="height:42px;width:228px;">Surface RNAse decontamination solution</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">AM9780</td>
			<td>RNaseZap</td>
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			<td height="21" style="height:21px;width:228px;">Fixative</td>
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			<td>70% Ethanol kept at -20 &#176;C</td>
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			<td height="21" style="height:21px;width:228px;">Anti-TH&#160;</td>
			<td style="width:148px;">Pel-Freez</td>
			<td style="width:99px;">P40101</td>
			<td>&#160;1:25</td>
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			<td height="21" style="height:21px;width:228px;">RNAse free buffer</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td>DEPC PBS + 1% BSA+0.02% triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNAse inhibitor</td>
			<td style="width:148px;">Promega</td>
			<td style="width:99px;">N2615</td>
			<td>Rnasine Plus Rnase Inhibitor 40 kU/ml (stock)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Anti-rabbit biotinylated</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">BA-1000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Vectastain Elite ABC kit Standard</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">PK-6100</td>
			<td>8&#181;l/ml</td>
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			<td height="21" style="height:21px;width:228px;">DAB Peroxidase Substrate Kit&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">SK-4100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNA isolation kit</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">KIT0204</td>
			<td>Arcturus PicoPure RNA Isolation Kit</td>
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			<td height="25" style="height:25px;">H2O2 30%</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">HC4060</td>
			<td></td>
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			<td height="21" style="height:21px;width:228px;">Ethanol absolute</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Laser microdissection system</td>
			<td style="width:148px;">Leica microsystems</td>
			<td style="width:99px;"></td>
			<td>Model AS-LMD</td>
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			<td height="21" style="height:21px;width:228px;">DEPC</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">B22753-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:99px;"></td>
			<td>Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Embedding mold&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">14702218311</td>
			<td>6x8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Hydrophobic barrier pen&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Frozen tissue embedding media</td>
			<td style="width:148px;">Tissue-Tek</td>
			<td style="width:99px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer chip</td>
			<td style="width:148px;">Aligent Technologies</td>
			<td style="width:99px;">5067-1513</td>
			<td>RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Hot start reaction mix for quantitative PCR</td>
			<td style="width:148px;">Roche</td>
			<td style="width:99px;">6402712001</td>
			<td>Fast Start Essential DNA Green Master</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Reverse transcriptase</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">11752-050</td>
			<td>SuperScript III First-Strand Synthesis SuperMix for qRT-PCR</td>
		</tr>
	</tbody>
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rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling

Laser capture mikrodissektion (LCM) muliggør isolering af specifikke celler fra tynde vævssnit med høj rumlig opløsning. Effektiv LCM kræver præcis identifikation af celler subpopulationer fra en heterogen væv. Identifikation af celler af interesse for LCM er normalt baseret på morfologiske kriterier eller fluorescerende protein journalister. Kombinationen af ​​LCM og hurtig immunolabeling tilbyder et alternativ og effektive midler til at visualisere bestemte celletyper og isolere dem fra omgivende væv. Høj kvalitet RNA kan derefter udvindes fra en ren cellepopulation og viderebehandles for downstream-applikationer, herunder RNA-sekventering, microarray eller QRT-PCR. Denne fremgangsmåde er tidligere blevet udført, og kort beskrevet i nogle publikationer. Målet med denne artikel er at illustrere, hvordan du udfører hurtig immunolabeling af en celle population samtidig holde RNA integritet, og hvordan man isolere disse specifikke celler ved anvendelse LCM. Heri vi illustrered denne multi-trins procedure immunolabeling og opfange dopaminerge celler i hjernevæv fra en dag gamle mus. Vi fremhæve vigtige kritiske skridt, der fortjener særlig opmærksomhed. Denne protokol kan tilpasses til en række af væv og celler af interesse. Forskere fra forskellige områder vil sandsynligvis drage fordel af demonstration af denne fremgangsmåde.

Denne hurtige immunolabeling procedure tillader visualisering af celler af interesse og samtidig holde RNA integritet. Figur 1 illustrerer trinnene forberedelse væv før RNA-ekstraktion. Efter cryosectioning er dopaminerge neuroner mærket under anvendelse af et antistof rettet mod tyrosin hydroxylase (mærkede neuroner vises i brun). Afhængig af den eksperimentelle spørgsmål, kan enkelte celler eller større område af interesse isoleres ved anvendelse af LCM (figur 1D - E)...

Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Genekspressionsanalyse af en delmængde af celler i et specifikt væv er en stor udfordring. I denne artikel beskrives, hvordan du isolere høj kvalitet total RNA fra en bestemt celle population ved at kombinere en hurtig immunolabeling metode med laser capture mikrodissektion.

RNA Isolation fra celle specielle befolkningsgrupper ved hjælp af laser-capture mikrodissektion Kombineret med Rapid immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

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