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.).

endoskopik retrograd kolanjiopankreatografi ile hastaların safra elde edilmesi (ERCP) Kurumsal Değerlendirme Kurulu tarafından bir insan denekler çalışma protokolünün onaylanması gerekiyor. Burada sunulan tüm çalışmalar Johns Hopkins Üniversitesi Kurumsal Değerlendirme Kurulu tarafından onaylandı. Bile 1. Exosome İzolasyon <ol><li> endoskopik retrograd kolanjiyopankreatografi (ERCP) gerçekleştirin. yatar pozisyonda hastayı ve entübe. <ol><li> Hastanın ağız yoluyla ...

<ol>
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güvenilir safra izole eksozomlar yararlanmak için, karşılığında yüksek kaliteli örnekleri elde etmek için tutarlı, yüksek kaliteli izolasyon yöntemleri kullanılması önemlidir. Bu yazıda tanımlanan metodoloji, insan safra gelen eksozomlar ve miRNA izole etmek köklü bir yoldur. Bu en azından elektron mikroskobu ya da nanoparçacık optik analiz ve Western lekeleri içermelidir izole Eksozomlann karakterizasyonu birkaç önemli adımlar vurgulamaktadır. o miRNA istikrar ge...

Diğer biyolojik yani sıvıların, anne sütü, plazma veya idrar gibi, safra ekzozomlar, lipit zengin veziküller 1-4 içerir. Eksozomlar neden veya alıcı hücreler 5, 6, normal fonksiyon 4, 7-9 bir parçası olabilir, hücre-hücre iletişiminin bir biçimde biyolojik fonksiyonları değiştirebilir. Eksozomlar tanı 10 biyomarkerların değerli bir kaynak sağlayabilir miRNA türlerini içerebilir. miRNA&#39;lar profilleri hastalıkların 11-14 çeşitli tanısal ve prognostik belirteç olarak son yıllarda önemli önem kazanmıştır. miRNA kendisi biyolojik sıvılarda, muhtemelen ölü hücrelerden salınan ve döken hücrelerin miktarı büyük ölçüde altta yatan hastalığa etkilemiş olabilir bulunabilir. Eksozom miRNA profili mutlaka kaynak hücresine 6, 10, 15-17 miRNA profili yansıtmaz, henüz ekzozomlar ebeveyn ce için karakteristik olabilir miRNA imzaları taşıyabilirBir tümör hücresi gibi olacağım. Tümör kaynaklı eksozomlar, akciğer adenokarsinom, prostat kanseri ve diğer tümörlerin 8, 16, 18 olan hastaların plazmasında belirlenmiştir. Bu yöntemin amacı, onların önceki kullanım prosedürleri büyük ölçüde bağımsız insan safra örneklerinden gelen eksozomlann güvenilir ve sağlam izolasyon olduğunu. Böyle kolanjiokarsinomlar 19 olarak safra kanalının hastalıkların potansiyel tanısı için miRNA güvenilir bir kaynak olarak safra kullanmak için geliştirilmiştir. Bu eksozomlar izole etmek hızları artan santrifüj birkaç aşamadan oluşmaktadır ve diğer biyolojik sıvılarda uygulanabilir olabilir.

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			<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>
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isolation and profiling of microrna-containing exosomes from human bile

son üç yılda exosome araştırma büyük ölçüde biyomarkerların ve bunların terapötik kullanımları tanımlanması ve karakterizasyonu doğru kapsamını genişletmiştir. Eksozomlar en son microRNA (MIRS) içerdiği gösterilmiştir. MIRS kendileri tanısal amaçlı değerli biyomarkerların olarak ortaya çıkmıştır. klinik ve hastanelerde numune toplama oldukça değişken olduğundan, tüm safra gelen miRNA izolasyon önemli ölçüde değişir. basit bir şekilde toplanan safra örnekleri, sağlam, güvenilir ve tekrar MiRNA profilleri elde etmek için izole etmek ve safradan eksozomlar karakterize etmek için yüksek kaliteli bir protokolünün geliştirilmesi gerekmektedir. yöntem, birkaç santrüfüj ve izole eksozomlar pelet nihai ultrasantrifügasyon adım bir filtre etme adımı gerektirir. Elektron mikroskobu, Western blot, flow sitometri ve çok parametreli nanoparçacık optik analiz, mevcut, e kalitesini doğrulamak için çok önemli karakterizasyon adımlardırxosomes. Örneğin Caenorhabditis elegans gibi bir türden gelen bir spesifik olmayan sentetik miRNA&#39;nın ile lisat spike bu eksozom Mirna izolasyonu için, Cel-miR-39, RNA ekstraksiyon verimi normalleştirilmesi için önemlidir. Bu yöntem aşağıdaki safra sıvısından eksozom izolasyonu başarılı MiRNA -80 ° C&#39;de birkaç yıl boyunca saklanabilir safra örnekleri profil sağlar.

eksozomlar normal mikroskobu ile tespit ya da akış sitometrisi için çok küçük olduğu için, elektron mikroskopisi ya da optik nanopartikül analizi gerçekleştirilmelidir. nanoparçacık optik analizi, aynı zamanda niceliksel ve boyutu dağılımına ve konsantrasyonu sağlar elektron mikroskobu üzerinde bir avantaja sahiptir. alet numune içine cam prizmadan ince odaklanmış lazer ışını tanıttı. İzole veziküllerin Brown hareketi bir EMCCD yüksek hassasiyet kamera il...

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.

İzolasyon ve profil MicroRNA içeren İnsan Bile dan ekzozomlar

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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