一种改进沉淀法来隔离尿外来体
Summary
This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Corning 15ml Centrifuge Tube | Corning (Sigma Aldrich) | CLS430791 | |
| Protease Inhibitor Cocktail | Sigma Aldrich | P8340 | |
| Centrifuge | Sorvall | ||
| DL-dithiothreitol | Sigma Aldrich | D9779 | used at final concentration of 200 mg/ml |
| Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well | Invitrogen | NP0321BOX | For SDS-PAGE analysis |
| ECL Advance Western blotting detection kit | GE Healthcare Life Sciences | RPN2135 | For western blot detection of biomarkers |
| Exoquick-TC Reagent | System Biosciences (SBI) | EXOTC50A-1 | For exosome isolation |
| Exosome CD9 ELISA Complete Kit | System Biosciences (SBI) | EXOEL-CD9-A-1 | For exosome quantification |
| AllPrep DNA/RNA/Protein mini kit | Qiagen | 80004 | For DNA, RNA, Protein isolation |
| Rneasy MinElute Cleanup kit | Qiagen | 74204 | |
| NanoDrop 2000 UV-Vis Spectrophotometer | Thermo Scientific | For Protein Quantification | |
| ProteoSilver Sliver Stain Kit | Sigma Aldrich | PROTSIL1-1KT | For staining SDS-PAGE gels |
| Xcell SureLock Mini-Cell Electrophoresis System | Life Technologies | For running the SDS-PAGE gels | |
| TaqMan microRNA assays | Life Technologies | For RT-PCR assays | |
| qBasePlus v1.5 software | Biogazelle NV | For analysis of RT-PCR assay data | |
| Rabbit polyclonal antibody to ALIX | Millipore | For western blot detection of biomarkers | |
| Mouse monoclonal antibody to TSG101 | Abcam | For western blot detection of biomarkers |
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