We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.

注意：涉及采用改良沉淀法的尿中外来体的分离的步骤示于图1中的初始步骤涉及去除大的死细胞和细胞碎片通过离心。从每个后续步骤所收集的上清液中得到的最终沉淀对应于外来体，将其溶于蛋白质，RNA和DNA的进一步萃取分离溶液。 1.收集尿<ol><li>之前收集尿液，根据制造商的说明制备蛋白酶抑制剂混合物，并添加3.125毫升此鸡尾酒到空的无菌容器中。含有蛋白酶抑制剂的管子就可以分发到志愿者尿液收集。 注：之所以添加蛋白酶抑制剂，是尽量减少尿样中蛋白降解。虽然我们专门用一种蛋白酶抑制剂的鸡尾酒西格玛，其它市售的蛋白酶抑制剂可以被取代。 </li><li>收集大约10毫升第一无效尿液。处理尿不储存或冷藏收集后立即使用。在隔离使用修改后的沉淀法重复尿外来体;的重复中的一个是用于DNA，总RNA和蛋白质的提取，而另一种是对泌尿外来体颗粒的定量。 注：24小时尿样可以通过存储在4℃可收集切体隔离。 </li></ol> 2.去除细胞碎片和TH糖蛋白（THP） <ol><li>传送尿液样本到15ml离心管中。离心机将10ml的尿液在17000×g离心10分钟，在37℃下，以消除细胞和细胞碎片（ 图1）。 </li><li>使用移液管，将上清转移到新的管中，以便进一步处理。重悬在500μl的隔离溶液的粒料（250毫蔗糖，10mM的熬炼anolamine，pH值7.6），接着用10分钟温育与DL-二硫苏糖醇（DTT：终浓度200毫克/毫升），在37℃。 注：DTT加到降解的THP，该捕获外来体，导致降低产率。照顾留上清液转移期间不受干扰的沉淀，并确认上清液是清澈粒料。 </li><li>涡旋药丸样品每2分钟，直到沉淀完全溶解。然后加入500μl的隔离溶液溶解的颗粒。 </li><li>紧接着加入隔离解决了沉淀，离心机在17000×g离心10分钟，在37℃。吸管将上清液与结合早期收集上清液。 </li><li>重悬在50μl的SDS-PAGE分析分离溶液中的沉淀。 注：PBS或TBS，可以用来代替分离溶液，以悬浮颗粒。 </li></ol> 3.隔离外来体颗粒的<ol><li>到11毫升收集supern的atant，添加3.3 ExoQuick-TC试剂中并孵育在4℃下至少12小时。 注：上清液ExoQuick-TC试剂的体积的比例可以进行调整，以增加或减少外来体的产率。我们观察到，较小的体积加入试剂生产外来体的小产量。 </li><li>培养后，离心样品/ ExoQuick-TC的混合物在10,000rpm×g离心30分钟，在25℃下。 </li><li>将上清液转移到新的管中进行SDS-PAGE分析。 </li><li>外来体粒料可以储存在-80℃用于DNA，RNA和蛋白质的提取，并使用酶联免疫吸附CD9 11外来体颗粒的定量。 注：外来体粒料可以储存在-80℃为1年有限冻融。 </li></ol> 4.生物标志物检测和外切体颗粒分析<ol><li>对于下游分析，使用DNA / RNA /蛋白质分离试剂盒和RNA分离き提取​​DNA，RNA和蛋白质的外来体沉淀根据制造商的说明吨。通过测定吸光度，在260和280nm处具有2微升样品的测定使用NanoDrop分光光度计浓度。 </li><li>进行实时定量RT-PCR（定量PCR）荧光定量使用的microRNA检测（在本例中，我们使用人类的miR-192和miR-1207-5p专用检测）或miRNA特异引物。 </li><li>对于SDS-PAGE分析，使用4-12％的Bis-Tris凝胶和分离蛋白质样品通过一维电泳。可视化，根据制造商的说明使用银染色试剂盒凝胶。 </li><li>执行根据由制造商提供的说明使用PVDF膜免疫印迹分析。使用兔多克隆抗体细胞凋亡连锁基因-2相互作用蛋白X或ALIX和鼠单克隆抗体肿瘤易感基因101蛋白或TSG101在免疫印迹检测。通过开放获取NIH ImageJ的软件量化带的密度（http://rsbweb.nih.gov/ij/）或一个类似的计划。 </li><li>量化用CD9 ELISA试剂盒按照制造商的说明获得的尿中外来体颗粒的数目。 </li></ol>

<ol>
	<li>Miranda, K. C., Bond, D. T., McKee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acids+within+urinary+exosomes/microvesicles+are+potential+biomarkers+for+renal+disease.">Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.</a> Kidney Int. 78, 191-199 (2010).</li><li>Pisitkun, T., Shen, R. F., Knepper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+proteomic+profiling+of+exosomes+in+human+urine.">Identification and proteomic profiling of exosomes in human urine.</a> Proc Natl Acad Sci USA. 101, 13368-13373 (2004).</li><li>Raj, D. 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大多数涉及外来体的沉淀，从尿方法不涉及脱除TH糖蛋白（THP）形成的聚合网络。这种蛋白是大量存在于尿液，其中在初始低速离心它推定陷阱外来体。在我们改进的方法，我们减少THP之前使用DTT以切体沉淀。 如图2中 ，观察到了更高量的THP在未处理的尿和沉淀，并在最后的上清液和外来体沉淀无THP，表明除去蛋白质。蛋白中的球团的量化表示6倍更高的产率，并且在沉淀的CD9的ELISA定...

尿外来体是尿空间正常和病理条件下，包括肾小球足细胞，肾小管细胞和细胞衬里，似乎是富集的miRNA 1的尿液引流系统下，来自所有类型的细胞多泡体（MVB）100nm的小囊泡内-2。许多研究人员已经发现不同的蛋白质在外来体从尿健康个体以及那些有肾和/或系统性疾病3-5隔离。外来体可以使用不同的方法，如两步骤差动超速离心有或无蔗糖6-7分离，结合纳米薄膜过滤器与超速离心8-9，或沉淀10-11。我们最近开发出一种简单，快速，可扩展，有效的方法来隔离尿外来体和检测miRNA与蛋白质生物标志物11。虽然生物标志物可以以各种不同的生物流体的检测出来，乌尔INE是患者的，因为它可以大量在相对非侵入性的方式获得的肾和泌尿道疾病的最方便的选择。 虽然有几种方法以分离尿液外来体6-11，最常用的方法依赖于微分超速离心用30％的蔗糖垫。虽然这种方法是有效的，并分离出与此过程所产生的外来体的质量是高的，该技术本身需要熟练的人员，是费时的，需要几个超速离心步骤。其他的方法，如过滤和沉淀，已经开发了用于外来体的分离，其中包括一个使用一个专有的沉淀试剂（ 即 ExoQuick-TC：系统Biosciences公司;加州山景城）。虽然用这个试剂沉淀是快速和相对容易的，相比于超速离心甲基分离的外泌体的纯度低OD。我们最近比较六种方法尿外来体的分离，包括使用ExoQuick-TC，我们开发了在我们的实验室11内的沉淀法的变形例。我们发现，这种改性沉淀法产生更高量的蛋白质，的miRNA和mRNA的并且过程本身是更快和更容易实现比超速离心。在这里，我们描述了在一个逐步​​的方式修改后的沉淀法的实验方案，并包括这种技术与外来体分离的其他方法相比的优点和缺点的讨论。改性沉淀方法包括外来体颗粒的初始沉淀，然后除去TH糖蛋白，它是相关与外来体蛋白，以及已知的，以减少从尿液外来体的产率。我们发现，这些修饰增加了从尿液外来体制剂的产率和纯度。

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 15ml Centrifuge Tube</td>
			<td style="width:111px;">Corning (Sigma Aldrich)</td>
			<td style="width:119px;">CLS430791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease Inhibitor Cocktail</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:111px;">Sorvall</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-dithiothreitol</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">D9779</td>
			<td>used at final concentration of 200 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>For SDS-PAGE analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL Advance Western blotting detection kit</td>
			<td style="width:111px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2135</td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exoquick-TC Reagent</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOTC50A-1</td>
			<td>For exosome isolation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exosome CD9 ELISA Complete Kit</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOEL-CD9-A-1</td>
			<td>For exosome quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AllPrep DNA/RNA/Protein mini kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">80004</td>
			<td>For DNA, RNA, Protein isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>For Protein Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProteoSilver Sliver Stain Kit</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">PROTSIL1-1KT</td>
			<td>For staining SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xcell SureLock Mini-Cell Electrophoresis System</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For running the SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan microRNA assays</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For RT-PCR assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qBasePlus v1.5 software</td>
			<td style="width:111px;">Biogazelle NV</td>
			<td style="width:119px;"></td>
			<td>For analysis of RT-PCR assay data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal antibody to ALIX</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse monoclonal antibody to TSG101</td>
			<td style="width:111px;">Abcam</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
	</tbody>
</table>

a modified precipitation method to isolate urinary exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

尿外来体进行肾上皮细胞分泌的蛋白质，miRNA与mRNA的生物标志物。这些外来体的分离和检测可以提供用于早期检测肾脏疾病的有用的诊断工具，例如糖尿病性肾病。有几种方法用于从人受试者收集的尿液样本的外来体的隔离。我们以前相比，尿外来体分离六种方法，调查得到的蛋白质的mRNA和miRNA水平11。这里我们的目标是在详细描述我们已经开发了用于尿外来体的高效分离改性沉淀法。 </...

Watch this Scientific Journal Video about A Modified Precipitation Method to Isolate Urinary Exosomes at JoVE.com

A Modified Precipitation Method to Isolate Urinary Exosomes

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

a-modified-precipitation-method-to-isolate-urinary-exosomes

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JoVE Journal

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17.5K 次观看.  Translational Genomics Research Institute (TGen). This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.