Name: isolation and profiling of microrna-containing exosomes from human bile
Uploaded: 2016-06-13T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

Ottenere la bile da pazienti di colangiopancreatografia retrograda endoscopica (ERCP) richiede l&#39;approvazione di un protocollo di studio soggetti umani dal Institutional Review Board. Tutto il lavoro qui presentato è stato approvato dal Comitato Etico della Johns Hopkins University. 1. Exosome Isolamento da Bile <ol><li> Eseguire colangiopancreatografia retrograda endoscopica (ERCP). Posizionare il paziente in posizione supina e intubare. <ol><li> Passare una duodenoscopio attraverso ...

<ol>
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Per utilizzare in modo affidabile esosomi isolati dalla bile, è importante utilizzare metodi di isolamento di alta qualità costante per ottenere campioni di alta qualità in cambio. La metodologia definita in questo documento è un modo consolidato per isolare esosomi e miRNA dalla bile umana. Si evidenzia diversi passaggi cruciali nella caratterizzazione dei esosomi isolati che come minimo dovrebbe comprendere microscopia elettronica o l&#39;analisi ottica di nanoparticelle e macchie occidentali. <p class="jove_c...

Come altri fluidi biologici, vale a dire, il latte materno, plasma o urina, la bile contiene esosomi, lipidi vescicole ricche 1-4. Esosomi possono indurre o alterare le funzioni biologiche in cellule riceventi 5, 6, una forma di comunicazione cellula-cellula che potrebbe essere parte della loro normale funzione 4, 7-9. Esosomi possono contenere specie miRNA che possono fornire una preziosa fonte di biomarcatori per la diagnosi 10. profili miRNA hanno guadagnato considerevole attenzione negli ultimi anni come marcatori diagnostici e prognostici per una varietà di malattie 11-14. miRNA stesso può essere trovato in fluidi biologici, eventualmente rilasciati da cellule morte e la quantità di cellule capannone può essere fortemente influenzato da una malattia di base. Il profilo di miRNA exosomes non riflette necessariamente il profilo miRNA della cellula originaria 6, 10, 15-17, ma exosomes può effettuare firme miRNA che potrebbero essere caratteristico per la ce genitorill come una cellula tumorale. Exosomes tumore-derivato sono stati identificati nel plasma dei pazienti con adenocarcinoma del polmone, cancro alla prostata e di altri tumori 8, 16, 18. L&#39;obiettivo di questo metodo è l&#39;isolamento affidabile e robusta di esosomi da campioni bile umana, in gran parte indipendenti delle loro procedure di gestione precedenti. E &#39;stato sviluppato per utilizzare la bile come una fonte affidabile di miRNA per il potenziale diagnosi delle malattie del dotto biliare, come colangiocarcinoma 19. Si compone di diverse fasi di centrifugazione con l&#39;aumentare della velocità per isolare esosomi e potrebbe essere applicabile ad altri fluidi biologici.

<table border="0" cellpadding="0" cellspacing="0" width="752">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">0.2&#181;m Polyethersulfone (PES) membrane filter</td>
			<td style="width:161px;">Corning</td>
			<td align="right" style="width:119px;">431229</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thinwall polyallomer ultracentrifuge tubes&#160;</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">331374</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">SW40Ti rotor</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">LM10-HS&#160;</td>
			<td style="width:161px;">Nanosight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Nanoparticle Tracking Analysis (NTA) software&#160;</td>
			<td style="width:161px;">Nanosight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mirVANA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">AM1560</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">cOmplete Protease Inhibitor</td>
			<td style="width:161px;">Roche distributed by Sigma-Aldrich</td>
			<td align="right" style="width:119px;">4693116001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Immobilon PSQ</td>
			<td style="width:161px;">Millipore, Bedford MA</td>
			<td style="width:119px;">ISEQ00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Anti-CD63 antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab59479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Anti-Tsg101</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab30871</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and profiling of microrna-containing exosomes from human bile

la ricerca Exosome negli ultimi tre anni ha notevolmente ampliato la portata verso l&#39;identificazione e la caratterizzazione di biomarcatori e loro usi terapeutici. Esosomi sono stati recentemente dimostrato di contenere microRNA (miR). Mirs si sono sorti come biomarcatori preziosi per scopi diagnostici. Come la raccolta di campioni nelle cliniche e negli ospedali è molto variabile, miRNA isolamento da tutta la bile varia notevolmente. Per ottenere solidi, precisi e riproducibili profili miRNA da campioni di bile raccolti in modo semplice richiesto lo sviluppo di un protocollo di alta qualità per isolare e caratterizzare esosomi dalla bile. Il metodo richiede diversi centrifugazioni e una fase di filtrazione con un passo ultracentrifugazione finale per far sedimentare le esosomi isolati. La microscopia elettronica, Western blot, citometria a flusso e multi-parametro analisi ottica di nanoparticelle, se disponibili, sono passi fondamentali di caratterizzazione per convalidare la qualità del messaggio exosomes. Per l&#39;isolamento di miRNA da questi esosomi, chiodare il lisato con un non-specifica, miRNA sintetico da una specie come Caenorhabditis elegans, vale a dire, Cel-miR-39, è importante per la normalizzazione di efficienza di estrazione di RNA. L&#39;isolamento di exosome dal liquido biliare seguendo questo metodo consente la riuscita miRNA profilatura da campioni di bile conservati per diversi anni a -80 ° C.

Poiché esosomi sono troppo piccole per essere rilevato mediante microscopia normale o la citometria a flusso, microscopia elettronica o analisi ottica nanoparticelle deve essere eseguita. L&#39;analisi ottica nanoparticelle ha il vantaggio rispetto microscopia elettronica che è anche quantitativa e fornisce distribuzione dimensionale e concentrazione. Lo strumento presenta un raggio laser finemente focalizzato attraverso un prisma di vetro nel campione. Il moto browniano delle vescicol...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

The overall goal of this method is to isolate exosomes from human bile for further analyses including microRNA profiling. This method can help answer key questions in the liver disease field such as detection of a particular bile marker for cholangiocarcinomas or other biliary diseases. The main advantage of this technique is that this is robust and repeatable.To perform endoscopic retrograde cholangiopancreatography, or ERCP, begin by placing the patient in a supine position. After intubating and passing a duodenoscope through the mouth of the patient, insert it into the second part of the duodenum and identify the major papilla. Selectively cannulate the common bile duct by inserting a triple lumen sphincterotome preloaded with a 0.35-inch or a 0.9-millimeter hydrophilic guidewire.Under fluoroscopic guidance, advance the guidewire to the left hepatic duct then advance the sphincterotome five centimeters into the bile duct to acquire a stable position. After removing the guidewire, attach a 10-millimeter syringe to the guidewire port through the lower lock and use 10 milliliters of negative pressure to aspirate the bile. Remove the syringe containing the bile and after reinserting the guidewire, inject contrast agent through the injection port to opacify the biliary tree.Then, transfer the collected bile into a 15-milliliter tube on ice and if storing for a later time, centrifuge the sample at 500 times g and four degrees Celsius for 10 minutes before freezing at minus 80 degrees Celsius. To immediately proceed with exosome isolation, transfer 400 microliters of bile into a microcentrifuge tube and centrifuge at 300 times g and four degrees Celsius for 10 minutes to collect cells and cell debris. To further clear the supernatant, transfer the solution to fresh microcentrifuge tubes and centrifuge the samples at 16, 500 times g and four degrees Celsius for 20 minutes.Then in a biosafety cabinet, collect the supernatant into a syringe and filter it through a 0.2-micrometer polyethersulfone membrane filter to remove any remaining particles larger than 200 nanometers. Transfer the filtered supernatant to ultracentrifuge tubes and use PBS to bring the tubes up to at least two-thirds full to prevent the tubes from collapsing during centrifugation. Pellet the exosomes through ultracentrifugation at 120, 000 times g and four degrees Celsius for 70 minutes.A small, yellow pellet will contain the exosomes. Discard the supernatant by carefully decanting and disinfect the waste by adding bleach to a final concentration of 10%volume per volume. Then let it stand for 20 minutes.For downstream experiments, either process the pellets in a small volume of appropriate solution or re-suspend them in 50 to 100 microliters of PBS and store at minus 80 degrees Celsius for future use. After preparing grids of contrast-stained extracellular vesicles, or EVs, according to the text protocol, acquire images with an electron microscope using an acceleration voltage of 80 kilovolts starting at magnifications of 20, 000 X, and increasing to 100, 000 X, when determining the size of the particles. Exosomes are too small to be detected by regular microscopy or flow cytometry;however, this figure shows the results of a typical nanoparticle tracking analysis, or NTA, of exosomes that measure the concentration and size distribution, which averaged 97 nanometers in this isolated human bile sample.Electron microscopy can also be used to confirm the size of the isolated particles in human bile and this figure shows a typical transmission EM result. These western blots were probed for proteins enriched in exosomes such as the tetraspanin CD63 and Tsg101. This real-time amplification plot shows a variety of microRNA species extracted from exosomes of human bile.Since exosomes lack reliable standards like 18S or 28S ribosomal RNA or housekeeping genes, the spiking of a synthetic microRNA is important for normalization. the exos isolation technique can be done in three hours if it is performed properly. After watching this video, you should have a good understanding of how to obtain and process bile from patients or animals to isolate and analyze extracellular vesicles.While recanting this procedure, it is important to remember to centrifuge the sample at 500 G and four degrees before freezing at minus 80, if you can't proceed with ultracentrifugation. Following this procedure, other methods like microRNA studies can be performed in order to answer additional questions like microRNA profiles. Don't forget that working with bile can be extremely hazardous since it could contain hepatitis, and precautions such as wearing gloves and safety glasses should always be taken while performing this procedure.After its development, the technique paved the way for research in the field of cholangiocarcinoma research to explore extracellular vesicles as potential biomarker in bile from patients with biliary problems such as obstruction of PSA.

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