Name: microrna in situ hybridization for formalin fixed kidney tissues
Uploaded: 2013-11-30T19:30:00
Description: Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

Dette arbejde blev støttet af US National Institutes of Health tilskud HL082798 og HL111580.

1.. Nyrevævssnittene <ol><li> Formalin-fikseret (10%) paraffinindstøbte nyresnit skæres over på objektglas ved 5 mm tykkelse ved hjælp af en microM HM 355 S. slides kan opbevares i adskillige måneder før analyse. </li></ol> 2. Solution Preparation <ol><li> Forbered 1 liter 1x PBS i ddH2O i en autoklaverbar skruelåg flaske. Fyld en anden flaske med 1 L af Hedeselskabet 2 O. Der tilsættes 1 ml DEPC til hver flaske, låg og ryst kraftigt. Lad løsninger sidde natte...

<ol>
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G., Ferracin, M., Calin, G. A., Fassan, M., Bassi, P., Sevignani, C., Byrne, D., Negrini, M., Pagano, F., Gomella, L. G., Croce, C. M., Baffa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-RNA+profiling+in+kidney+and+bladder+cancers.">Micro-RNA profiling in kidney and bladder cancers.</a> Urol. Oncol. 25 (5), 387-392 (2007).</li><li>Juan, D., Alexe, G., Antes, T., Liu, H., Madabhushi, A., Delisi, C., Ganesan, S., Bhanot, G., Liou, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+microRNA+panel+for+clear-cell+kidney+cancer.">Identification of a microRNA panel for clear-cell kidney cancer.</a> Urol. 75 (4), 835-841 (2010).</li><li>Xu, X., Kriegel, A. 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Målet med denne artikel var at beskrive en protokol for miRNA in situ hybridisering, der fungerer godt i formalin faste nyrevæv. Mens de arbejder ud denne protokol flere vigtige kilder til farvning artefakt er blevet identificeret. Omhyggelig opmærksomhed på disse punkter kan bidrage til at undgå pletter artefakt og øge sandsynligheden for en vellykket ISH løb. En af de mest undgåelige årsager til farvning artefakt kan opstå, når vævssnit blevet tørret ud i lange inkuba...

MicroRNA er små (ca. 22 nukleotider lange) noncoding RNA, der er endogent produceret. De fungerer generelt at undertrykke protein udtryk gennem translationel undertrykkelse eller mRNA nedbrydning. miRNA binder til mRNA-mål med ufuldstændig komplementaritet, hvilket gør det muligt for en enkelt miRNA at undertrykke flere mål. Forståelse som celletyper og strukturer udtrykker miRNA er en vigtig del af at forstå de mekanismer, hvorigennem ændringer i miRNA udtryk indflydelse celle og væv fænotyper. Mens metoder såsom miRNA sekventering, qPCR og Northern blotting kan anvendes til detektion af miRNA i hele væv, er denne fremgangsmåde ikke tillader en at bestemme, hvilken specifik celletype de kom fra inden for et givet væv. Dissektion af cellulære og strukturelle komponenter inden analyse ved hjælp af disse metoder kan være meget vanskeligt, og de betingelser, der er nødvendige for at opnå tilstrækkelige isolationer kan føre til alterations i genekspression eller forringelse af RNA. microRNA in situ hybridisering er en metode anvendt til at visualisere microRNA placering og ekspressionsniveauer i væv. Denne teknik er især værdifuldt i væv sammensat af heterogene strukturer, såsom nyrerne. MicroRNAs har vist sig at spille en regulerende rolle i nyre-udvikling 2,3 og fysiologi 4. har også vist microRNA udtryk ændringer for at blive involveret med nedsat patologi såsom fibrose 5-10, diabetisk nefropati 7, nyrekarcinom 11,12 og akut nyreskade 13. I vores forskning har vi fundet, at en optimering microRNA in situ hybridisering for nyrevæv var værdifuldt for fastsættelse af det præcise strukturelle placeringer af miRNA-ekspression i både sundhed og sygdom 14. Bestemmelse af det rørformede og cellulære udtryk for forskellige microRNA er vigtigt, fordi deres regulering af targets kan være afhængig af cellulære funktioner. I sygdomstilstande er det også vigtigt at bestemme, hvordan ændringer i miRNA ekspression kan påvirke funktionen. Målet med den her beskrevne fremgangsmåde var at bygge videre på eksisterende ISH metoder udviklet af Kloosterman et al. 15. andre efterforskere 16,17, og dem, der foreslås af Exiqon 1 og optimere metoden til formalin faste nyrevæv. Vi har med succes brugt denne metode til at identificere tydelige regionale forskelle i renal microRNA miR-382-ekspression med unilateral ureter obstruktion 18. Denne fremgangsmåde kan anvendes med andre væv samt, med yderligere optimering.

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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

I denne artikel beskriver vi en metode til kolorimetrisk detektion af miRNA i nyrerne gennem in situ hybridisering med digoxigenin tagged microRNA sonder. Denne protokol, der oprindeligt blev udviklet af Kloosterman og kolleger for bred anvendelse med Exiqon miRNA sonder 1, er blevet ændret til at overvinde udfordringer forbundet med miRNA-analyse i nyrevæv. Disse omfatter spørgsmål såsom identifikation struktur og svært at fjerne resterende sonde og antistof. Anvendelse af relativt tyndt, 5 mm tyk, vævssnit tilladt for klar visualisering af nyre-strukturer, mens en stærk sondesignal blev tilbageholdt i celler. Derudover blev probe koncentrations-og inkubationsbetingelser optimeret for at lette visualiseringen af ​​microRNA udtryk med lav baggrund og uspecifik signal. Her er optimeret beskrevne protokol, der dækker den indledende væv indsamling og forberedelse gennem montering af glider ved slutningen af ​​proceduren. Den grundlæggende componeNTS i denne protokol kan ændres til anvendelse til andre væv og cellekultur modeller.

De områder af et vævssnit, der bliver tørret i løbet af de hybridiseringer inkubationerne eller vaske trin generelt ender farvning mere mørkt i NBT / BCIP udvikling. Figur 1 viser en del af en nyre sektion, hvor HybriSlip gled ud af kanten af vævet, gør det muligt at blive delvist dehydreret. Trods rehydrering og dækning i de resterende trin, signalet i dehydreret delen er kunstigt høj. Betydningen af at styre tidspunktet for NBT-BCIP udvikling er demonstreret i <st...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Denne artikel beskriver en in situ hybridisering protokol optimeret til kolometriske detektion af microRNA udtryk i formalin faste nyresnit.

MicroRNA<em&gt; In situ</em&gt; Hybridisering for formalin Faste nyrevæv

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Radioaktivt In situ Hybridisering til påvisning af Forskellige genekspressionsmønstre i Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualisering og analyse af mRNA molekyler under anvendelse Fluorescens<em&gt; In Situ</em&gt; Hybridisering i<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Proteomiske Prøveforberedelse fra formalin Faste og Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

qPCR er en følsom og hurtig metode til påvisning af Cytomegaloviral DNA i formalin-faste, paraffinindstøbt biopsivæv

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...