分离和剖析人力胆汁含外来体的MicroRNA-
Summary
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.
Abstract
在过去的三年中外来体的研究，大大延长对生物标志物和治疗用途的识别和鉴定的范围。
外来体最近已显示含有微小RNA（miR的）。大鹏自己已经出现的用于诊断目的的宝贵的生物标志物。由于标本采集诊所和医院是变化很大，从整体胆汁miRNA分离差异很大。以实现从收集的胆汁样品可靠，精确和可重复的miRNA表达谱以简单的方式需要高品质的协议的发展，以分离和从胆汁表征外来体。该方法需要几离心并以最终超速离心步骤以沉淀分离的外泌体的过滤步骤。电子显微镜，Western印迹，流式细胞术和多参数纳米颗粒光学分析，其中，可用的，是至关重要的表征步骤来验证电子的质量xosomes。对的miRNA来自这些外来体的分离，从像线虫 ， 即物种扣球用非特异性的，合成的miRNA的裂解物，CEL-的miR-39，为的RNA提取效率正常化重要。外来体的从以下本方法胆汁流体隔离允许成功的miRNA从可保存数年在-80℃胆汁样品分析。
Introduction
像其他生物流体，即，母乳，血浆或尿液，胆汁中含有外来体，富含脂质的囊泡1-4。外来体可以诱发或在受体细胞5,6，细胞间通讯的一种形式，可能是它们的正常功能4，7-9的部分改变生物学功能。外来体可以包含miRNA族可诊断10提供的生物标志物的重要来源。微RNA型材近年来，作为多种疾病11-14诊断和预后标志物获得大量关注。
的miRNA本身可以在生物流体中，可能由死细胞释放和棚单元的量可以通过一个潜在的疾病受到很大影响被发现。外来体的miRNA谱并不一定反映原来的小区6，10，15-17的miRNA的轮廓，但外来体可以携带的miRNA签名，可能是父母的CE特点会喜欢肿瘤细胞。肿瘤来源的外来体已经患者肺腺癌，前列腺癌和其它肿瘤8，16，18在血浆中被确定。
该方法的目的是从人胆汁标本，很大程度上独立的其前处理程序的外来体的可靠和稳定的隔离。它的开发是为了利用胆汁作为的miRNA为的胆管的疾病，例如胆管癌19的电位诊断的可靠来源。它包括离心的几个步骤的增加速度以分离外泌体，并且可能适用于其他生物流体。
Protocol
Representative Results
Discussion
为了可靠地利用从胆汁中分离外来体，它是采用一贯的高品质分离方法的回报，以获得高质量的样本非常重要的。在本文中定义的方法，是从人胆汁隔离外来体和miRNA行之有效的办法。它强调在隔离外来其中至少应包括电子显微镜或纳米光学分析和蛋白质印迹的表征几个关键步骤。
在外来体的分离中最关键的一步，至少当它涉及到的miRNA的稳定性，就是要尽快处理新鲜胆汁。?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).
Materials
| 0.2µm Polyethersulfone (PES) membrane filter | Corning | 431229 | |
| thinwall polyallomer ultracentrifuge tubes | Beckman Coulter | 331374 | |
| SW40Ti rotor | Beckman Coulter | ||
| LM10-HS | Nanosight | ||
| Nanoparticle Tracking Analysis (NTA) software | Nanosight | ||
| mirVANA | Life Technologies | AM1560 | |
| cOmplete Protease Inhibitor | Roche distributed by Sigma-Aldrich | 4693116001 | |
| Immobilon PSQ | Millipore, Bedford MA | ISEQ00010 | |
| Anti-CD63 antibody | Abcam | ab59479 | |
| Anti-Tsg101 | Abcam | ab30871 |
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