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.).

L&#39;obtention de la bile de patients par cholangiopancréatographie rétrograde endoscopique (CPRE) nécessite l&#39;approbation d&#39;un protocole d&#39;étude sur des sujets humains par le Institutional Review Board. Tous les travaux présentés ici a été approuvé par le comité d&#39;examen institutionnel de l&#39;Université Johns Hopkins. 1. Exosome Isolation de Bile <ol><li> Effectuer cholangiopancréatographie rétrograde endoscopique (CPRE). Placer le patient en position couch...

<ol>
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Pour utiliser de manière fiable exosomes isolés de la bile, il est important d&#39;utiliser des méthodes d&#39;isolement de haute qualité compatibles pour obtenir des échantillons de haute qualité en retour. La méthodologie définie dans le présent document est un moyen bien établi pour isoler les exosomes et microARN de la bile humaine. Il met en évidence plusieurs étapes cruciales dans la caractérisation des exosomes isolés qui, à un minimum devrait comprendre la microscopie électronique ou optique de n...

Comme d&#39; autres fluides biologiques, à savoir, le lait maternel, le plasma ou l&#39; urine, la bile contient exosomes, lipides riches vésicules 1-4. Les exosomes peuvent induire ou altérer les fonctions biologiques dans les cellules réceptrices 5, 6, une forme de communication entre les cellules qui pourraient faire partie de leur fonction normale 4, 7-9. Les exosomes peuvent contenir des espèces miRNA qui peuvent fournir une source précieuse de biomarqueurs pour le diagnostic 10. les profils miARN ont gagné une attention considérable ces dernières années en tant que marqueurs diagnostiques et pronostiques pour une variété de maladies 11-14. miRNA lui-même peut être trouvé dans les fluides biologiques, peut-être libéré par les cellules mortes et la quantité de cellules de hangar peut être fortement influencée par une maladie sous-jacente. Le profil miRNA des exosomes ne reflète pas nécessairement le profil miRNA de la cellule d&#39;origine 6, 10, 15-17, mais exosomes peut porter des signatures miRNA qui pourraient être caractéristique de la CE parentalell aime une cellule tumorale. Exosomes dérivés de tumeur ont été identifiés dans le plasma de patients atteints d&#39; un adénocarcinome du poumon, le cancer de la prostate et d&#39; autres tumeurs 8, 16, 18. Le but de cette méthode est l&#39;isolement fiable et robuste des exosomes à partir d&#39;échantillons de bile humaine, largement indépendantes de leurs procédures de traitement antérieur. Il a été développé pour utiliser la bile en tant que source fiable d&#39;ARNmi pour le diagnostic potentiel des maladies du canal biliaire , tels que 19 cholangiocarcinome. Il se compose de plusieurs étapes de centrifugation, à des vitesses croissantes afin d&#39;isoler les exosomes et pourrait être appliquée à d&#39;autres fluides biologiques.

<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 recherche de Exosome au cours des trois dernières années, a considérablement étendu le champ vers l&#39;identification et la caractérisation de biomarqueurs et de leurs utilisations thérapeutiques. Il a été démontré récemment exosomes pour contenir microARN (MIRS). Mirs se sont apparus comme biomarqueurs de valeur à des fins de diagnostic. Comme le prélèvement d&#39;échantillons dans les cliniques et les hôpitaux est très variable, miRNA l&#39;isolement de la bile toute varie considérablement. Pour obtenir des profils de miARN robustes, précis et reproductibles à partir d&#39;échantillons de bile recueillies d&#39;une manière simple nécessité l&#39;élaboration d&#39;un protocole de haute qualité pour isoler et caractériser les exosomes de la bile. Le procédé nécessite plusieurs centrifugations et une étape de filtration par une étape finale d&#39;ultracentrifugation pour sédimenter les exosomes isolés. La microscopie électronique, Western blots, cytométrie en flux et l&#39;analyse optique nanoparticule multi-paramètres, le cas échéant, sont des étapes cruciales de caractérisation pour valider la qualité de l&#39;exosomes. Pour l&#39;isolement des miARN de ces exosomes, Smasher le lysat avec un miARN synthétique non spécifique d&#39;une espèce comme Caenorhabditis elegans, à savoir, Cel-miR-39, est important pour la normalisation de l&#39; efficacité d&#39;extraction d&#39;ARN. L&#39;isolement des exosomes à partir du liquide biliaire suivant cette méthode permet au succès miRNA profilage à partir d&#39;échantillons de bile stockées pendant plusieurs années à -80 ° C.

Etant donné que les exosomes sont trop petites pour être détectées par microscopie ordinaire ou cytométrie en flux, la microscopie électronique ou optique de nanoparticules doit être effectuée. L&#39;analyse optique de nanoparticules a l&#39;avantage par rapport à la microscopie électronique qu&#39;il est également quantitative et fournit une distribution granulométrique et la concentration. L&#39;instrument présente un faisceau laser finement focalisé à travers un prisme ...

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.

L&#39;isolement et le profilage des exosomes de Bile humain contenant microARN-

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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