This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

获取来自患者经内镜逆行胰胆管胆汁（ERCP）需要一个人类受试者的研究方案通过机构审查委员会批准。这里介绍的所有工作被批准为约翰·霍普金斯大学的机构审查委员会。 1.胆汁外来体隔离<ol><li>执行内镜逆行胰胆管造影（ERCP）。将患者仰卧位和插管。 <ol><li>传递十二指肠通过患者的口，并将其插入到十二指肠的第二个部分和确定主要乳头。 </li><li>选择性导管插入通过插入胆总管三腔sphincterotome预装一个0.035英寸（0.9 MM）亲水导丝。 </li><li>推进透视下导丝到左肝管，因为有时可能难以区分右肝管的胆囊管，然后前进sphincterotome5厘米到胆管获得一个稳定的位置。 </li><li>除去导丝，通过路厄锁连接10毫升注射器的导引线端口和吸用10ml负压的胆汁。 </li><li>删除包含胆汁通过重新插入导丝，并通过注入口注入造影剂变得不透明胆管树与ERCP进行注射器。 </li><li>传送所收集的胆汁入15ml试管在冰上，并保持在冰上直至准备与离心步骤来进行。 注意:戴上手套，以保护自己和样品！治疗胆汁感染的潜在来源，因为它可包含A型肝炎病毒颗粒！ </li><li>保持冰样本（S）。无论是与步骤1.2或离心机继续在500 xg离心在4℃下进行10分钟，然后在-80℃直至进一步使用冻结。 注:几年来以这种方式存储的样品已经被常规使用，效果很好19。 </li></ol&gt; </li><li>转移400μl的胆汁入微量离心管中并在4℃下以300 xg离心离心收集细胞和细胞碎片10分钟。 </li><li>通过移液除去上清液，转移到新的微量离心管中并在4℃以16500 XG离心样品（个），另外20分钟以清除进一步碎片的上清液。丢弃在生物危险废物管。可替代地，旋转样品中超速离心机代替。 </li><li>收集上清液并通过0.2微米的聚醚砜（PES）膜过滤器，它具有良好的流速和低蛋白结合以除去大于200nm左更大的任何颗粒在用注射器生物安全柜过滤。 </li><li>吸管过滤的上清液超速离心管并加入PBS中，使所述管超过​​2/3满。如果管含有比流动性较差，管将离心过程中崩溃！管可水洗，高压灭菌并重复使用多次。 </li><li>佩尔等通过超速离心的外来体以120,000lux xg离心70分​​钟，4℃。后离心看超速离心管到，有时会看到小黄粒料的底部;这些是外来体。 </li><li>通过小心倾析其弃去上清液，并通过加入漂白〜10％V / V的终浓度消毒废物，让它静置20分钟。 </li><li>下游的实验中，无论是处理在适当的溶液中的一小体积颗粒（多个）（ 即，放射免疫测定（RIPA）缓冲液用于蛋白分离，为的miR隔离的RNA裂解缓冲液），或在50-150微升磷酸盐悬浮它缓冲盐水（PBS），其可通过TEM或纳米颗粒的光学分析或存储在-80℃下以供将来使用（定量实时聚合酶链式反应（QRT-PCR）或蛋白质提取等 ）或用于外来体制备的表征。 </li></ol> 2.外来体鉴定透射电子显微镜（TEM）或CryoEM <ol><li>吹打重悬在100μlPBS中的细胞外囊泡。理想的是，使用还没有被保存在-20℃或-80℃的外来体，在第一次执行隔离时尤其如此。 </li><li>吸附20微升等分试样（约2微克）至400目碳包覆Parlodion铜网格为2分钟，并允许在室温下干燥。 </li><li>通过在干燥制剂小心放置滴固定外来体在1％（体积/体积）戊二醛（EM-级）在RT 5分钟。 </li><li>用水冲洗格两次5分钟，然后对比染色电动汽车用1％磷钨酸（PTA）30秒。 </li><li>与开始以20,000的放大率的80千伏的加速电压和确定颗粒的大小时，增大到100000的电子显微镜获得图像。 注意:像铺在福尔瓦碳包200目铜或镍网和对比染色Li的棉片科1％或2％乙酸双氧铀10-15分钟也是可能的。 </li><li> （3比1）CryoEM，用60微升蛋白G金10纳米混合20微升等份（约2微克）。这将有助于确定外来体的直径。 </li><li>转移到样品辉光放电C-平多孔碳电网和吸干除去多余的液体。通过立即浸渍他们到液态乙烷冻结网格。 </li><li>安装网格在液氮cryoholder。 </li><li>获得200千伏图像有20,000和100000的放大倍率。 </li></ol> 3.外来体鉴定多参数纳米光学分析<ol><li>吹打重悬在100μlPBS中的分离的外泌体。 </li><li>在几个不同的比率稀释在600μl的PBS中的外来体（如1:50 1:100 1:300 1:600和1:1200）。 </li><li>根据manufactu与实时适当仪器和软件可视用激光光散射的外来RER的协议20。 </li><li>增加相机的水平，捕捉模式下，直到颗粒变得可见，然后调节对焦，使粒子看起来光滑。 </li><li>而在标准模式下，减少相机的颗粒还是可以看到的最低水平，如果需要，调整相机的快门和增益来实现这一点。 </li><li>目视检查，以确保20-100颗粒在屏幕上可见，如果不刷新单元和调整PBS稀释。 </li><li>然后调整捕获持续时间30-60秒之间。视频捕获后，调节处理参数，直到所有的颗粒在屏幕上确定和处理该视频文件。 </li></ol> 4.外来体表征Western印迹<ol><li>从2-5在100μl冰冷的放射免疫测定法的胆汁中的溶液裂解外来体颗粒（RIPA）与蛋白酶抑制剂移液器向上缓冲和向下彻底和30秒剧烈混合裂解液上的涡流，有效地溶解蛋白质。 </li><li>而大部分时间没有必要的，在冰上用样品的脉冲超声处理用2个脉冲，每次10秒，以确保完全裂解。 </li><li>以15,000 xg离心沉淀通过离心不溶物在4℃下5分钟，转移上清液至干净的管中。 </li><li>与选择的方法，根据生产商的方案（ 即 ，二辛可宁酸（BCA），考马斯，改性的Lowry，660纳米蛋白测定等 ）确定蛋白质浓度。作为同一方法用于一致地实现可跨实验进行比较的结果的方法是没有限制，只要重要。 </li><li>负载在Laemmli缓冲每个样品的蛋白质的50微克，在95℃，5分钟加热样品到聚丙烯酰胺凝胶的孔为电泳（PAGE）后。 </li><li> Electrophorese在PAGE凝胶样品1.5小时，转移到尼龙膜上的分离的蛋白质。 </li><li>乙用0.1％吐温20（TBS-T）锁定用5％脱脂牛奶的Tris缓冲盐水的膜（个），1小时，然后在稀释像抗CD63和抗TSG-101特异性外来体抗体孵育1:500最好是O / N在4℃。 </li><li>洗用TBS-T 3×10分钟的膜，然后用合适的孵育，匹配HRP缀合的第二抗体在TBS-T中的5％脱脂牛奶在室温1-2小时。 </li><li>洗膜（多个）3×5分钟，用TBS-T和具有根据制造商的协议的化学发光试剂检测的特定蛋白质。 </li><li>图像与膜或根据制造商的协议的数字成像系统中的膜。 </li></ol> 5.从隔离外来体的MicroRNA隔离注意:从胆汁外来体的miRNA的分离是通过使用基于市售的试剂盒修饰的miRNA隔离，但也可以使用或适于任何其它的RNA分离方法。 <ol><li>添加300＃181:L裂解/建筑物缓冲器向外来体沉淀（从400μl的胆汁中分离），涡旋，吸管或穿过针/注射器以完全裂解外来体以获得均匀的溶胞产物。 </li><li>添加的miRNA匀浆添加剂的1/10（30微升）量，并通过涡旋或颠倒试管几次拌匀。 </li><li>孵育在冰上10分钟的混合。 </li><li>加酸 - 苯酚的体积:氯仿等于初始匀浆（300微升）。 </li><li>加入5飞摩尔/微升CEL-MIR 39的5微升，涡旋30-60秒。 </li><li>以10,000 xg离心在RT以分离含水（上）和有机（低级）相离心5分钟。 </li><li>通过移液和转移上层相小心吸至新管，同时注意到转移的体积。丢弃在适当的有机废物容器中的有机相。 </li><li>以丰富的小RNA体积的1/3 100％的乙醇添加到回收水相，通过混合涡旋彻底颠倒或涂数倍。 </li><li>吸管的裂解物/乙醇混合物到滤筒，高达700微升在一个时间。 </li><li>离心15秒的墨盒以最大万xg的或施加真空通过过滤器传递该混合物。 </li><li>收集滤液并且如果裂解物/乙醇混合物超过700微升，流过转移到新的管中，并重复收集所有滤液的过程。这些过滤器中包含的RNA级分不含小RNA和可用于回收这些较大的RNA。 </li><li>加2/3体积的RT 100％的乙醇的总滤液并通过涡旋调匀。 </li><li>吸管滤液/乙醇混合物到第二个过滤器滤芯。像之前可以一次施加的最大容积为700微升。 </li><li>离心15秒以10,000 xg离心或如在步骤5.10中所述施加真空。 </li><li>丢弃流过，并重复直到所有的滤液/乙醇混合物的已被过滤。 </li><li>应用70081，L的miRNA洗涤液1到滤筒，离心5-10秒或施加真空并丢弃流过。 </li><li>按照步骤完成5.16洗净用500微升洗涤液2/3的过滤器。 </li><li>重复洗涤第二次用500μl的洗涤溶液2/3如在步骤5.16，丢弃流过并且过滤器放回收集管。 </li><li>旋转滤筒1分钟以10,000 xg离心以从过滤器去除所有残余流体。 </li><li>滤筒转移到新鲜的收集管，并应用100微升的热（95℃）无核酸酶的水至过滤器的中心。 </li><li>旋转20-30秒的组件以最大速度收回RNA。 </li><li>收集洗脱液，并立即使用或在-80℃保存。 </li></ol>

<ol>
	<li>Admyre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+with+immune+modulatory+features+are+present+in+human+breast+milk.">Exosomes with immune modulatory features are present in human breast milk.</a> J. Immunol. 179 (3), 1969-1978 (2007).</li><li>Aushev, V. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+microRNA+patterns+in+plasma+before+and+after+tumor+removal+reveal+new+biomarkers+of+lung+squamous+cell+carcinoma.">Comparisons of microRNA patterns in plasma before and after tumor removal reveal new biomarkers of lung squamous cell carcinoma.</a> PLoS One. 8 (10), e78649 (2013).</li><li>Ben-Dov, I. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urine+microRNA+as+potential+biomarkers+of+autosomal+dominant+polycystic+kidney+disease+progression:+description+of+miRNA+profiles+at+baseline.">Urine microRNA as potential biomarkers of autosomal dominant polycystic kidney disease progression: description of miRNA profiles at baseline.</a> PLoS One. 9 (1), e86856 (2014).</li><li>Vlassov, A. V., Magdaleno, S., Setterquist, R., Conrad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+current+knowledge+of+their+composition,+biological+functions,+and+diagnostic+and+therapeutic+potentials.">Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.</a> Biochim. Biophys. Acta. 1820 (7), 940-948 (2012).</li><li>Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+mechanisms+and+intercellular+transfer+of+microRNAs+in+living+cells.">Secretory mechanisms and intercellular transfer of microRNAs in living cells.</a> J. Biol. Chem. 285 (23), 17442-17452 (2010).</li><li>Mittelbrunn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unidirectional+transfer+of+microRNA-loaded+exosomes+from+T+cells+to+antigen-presenting+cells.">Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.</a> Nat. Commun. 2, 282 (2011).</li><li>Pegtel, D. M., van de Garde, M. D., Middeldorp, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+miRNAs+exploiting+the+endosomal-exosomal+pathway+for+intercellular+cross-talk+and+immune+evasion.">Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion.</a> Biochim. Biophys. Acta. 1809 (11-12), 715-721 (2011).</li><li>Peinado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+exosomes+educate+bone+marrow+progenitor+cells+toward+a+pro-metastatic+phenotype+through+MET.">Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.</a> Nat. Med. 18 (6), 883-891 (2012).</li><li>Thery, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat. Rev. Immunol. 9 (8), 581-593 (2009).</li><li>Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J., Lotvall, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nat. Cell Biol. 9 (6), 654-659 (2007).</li><li>Bessho, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+genomics+identifies+candidate+microRNAs+for+pathogenesis+of+experimental+biliary+atresia.">Integrative genomics identifies candidate microRNAs for pathogenesis of experimental biliary atresia.</a> BMC Syst. Biol. 7, (2013).</li><li>Calin, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling+reveals+distinct+signatures+in+B+cell+chronic+lymphocytic+leukemias.">MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (32), 11755-11760 (2004).</li><li>Collins, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+microRNA+profile+distinguishes+cholangiocarcinoma+from+pancreatic+adenocarcinoma.">A differential microRNA profile distinguishes cholangiocarcinoma from pancreatic adenocarcinoma.</a> Ann. Surg. Oncol. 21 (1), 133-138 (2014).</li><li>He, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+polycistron+as+a+potential+human+oncogene.">A microRNA polycistron as a potential human oncogene.</a> Nature. 435 (7043), 828-833 (2005).</li><li>Taylor, D. D., Gercel-Taylor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+signatures+of+tumor-derived+exosomes+as+diagnostic+biomarkers+of+ovarian+cancer.">MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.</a> Gynecol. Oncol. 110 (1), 13-21 (2008).</li><li>Rabinowits, G., Gercel-Taylor, C., Day, J. M., Taylor, D. D., Kloecker, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+microRNA:+a+diagnostic+marker+for+lung+cancer.">Exosomal microRNA: a diagnostic marker for lung cancer.</a> Clin. Lung Cancer. 10 (1), 42-46 (2009).</li><li>Skog, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat.Cell Biol. 10 (12), 1470-1476 (2008).</li><li>Lance, R. S., Drake, R. R., Troyer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+recognition+assay+reveals+prostasomes+as+promising+plasma+biomarkers+for+prostate+cancer.">Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer.</a> Expert Rev. Anticancer Ther. 11 (9), 1341-1343 (2011).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bile+contains+microRNA-laden+extracellular+vesicles+that+can+be+used+for+cholangiocarcinoma+diagnosis.">Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.</a> Hepatology. 60 (3), 896-907 (2014).</li><li>Gabriel, D. A., Giordano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle+sizing+and+counting+using+light+scattering+methods.">Microparticle sizing and counting using light scattering methods.</a> Semin. Thromb. Hemost. 36 (8), 824-832 (2010).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu. Rev. Cell Dev. Biol. 30, 255-289 (2014).</li></ol>

None of the authors have competing financial interests.

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。在RT整个胆汁甚至单个冻融循环的延长存储可以显著降低的miRNA内容，而在相反，即使当储存在室温或经历几个冻融循环的分离的外泌体是非常稳定的。只要有问题的样本已被存储在-80℃下，外来体和miR的成功分离NA从胆汁可以以极大的可重复性进行，以鉴定样品中的miRNA特征。建议最初与新鲜胆汁开始，以确保适当的miRNA的完整性。这是试图在一段时间来建立胆汁样品的集合作为将是患者样品的情况下，当一个重要的考虑因素。它使人们能够处理已收集在数月或甚至数年的同时被处理和评价样品。 这极大地增强了使用胆汁用于诊断目的，无论是寻找该确认疾病状态如癌症的存在或疾病的治疗性干预的反应的miRNA签名。事实上，从临床样品的结果已被用于建立的miRNA特征作为生物标志物用于胆管癌19。 第一次离心步骤除去存在于胆汁完整细胞。在第二，更高的速度离心细胞DEBRIS，凋亡小体等细胞器更大的沉淀。为了确保只有小于200纳米在超速离心步骤中收集颗粒，具有低蛋白结合过滤器的过滤步骤是非常重要的。某些协议省略这一步，但我们认为这是必要的。以120,000lux XG超离心后获得的沉淀物包含既可以立即或再悬浮后处理在PBS贮存在-80℃相当纯外来体。这种方法的一个限制是，所得到的沉淀物只高度富集的外来体，但是这是用于其它富集方法诸如大小排阻和聚合沉淀如此。进一步的处理可能涉及由蔗糖梯度或结合抗体包被磁珠另一个纯化步骤。如果外来的来源（例如像等离子体）含有析出物作为凝血的结果，因为这些颗粒可通常具有外来体的大小这一额外纯化可能是必要的。特别是，免疫纯化导致一个更外来体标记富集制剂。的缺点是，这种进一步减少外来体和/或miRNA的存在量，对于大多数目的没有理由的耗费时间和资源的过程。然而，如果Western印迹分析检测微粒污染的通过内质网蛋白的存在下，如钙联接蛋白，进一步纯化，在基于特定免疫隔离，建议。在我们的经验，这是很少为胆汁的情况下，如果一个使用基于免疫纯化步骤将建议生物体液的其它来源。 使用差动超速离心来从胆汁流体分离和富集外来体是相对快速的和成本有效的方法。而它需要使用超速离心机的，最好用摆动斗转子，这些装置通常在许多机构中。通过密度离心进一步纯化是possible为相同的设备和管是清洁和消毒后可重复使用数次。而基于聚合物的沉淀只需要使用microcentrifuges或标准离心机以10,000 xg离心或更少的速度以沉淀析出物，通常共分离的非水泡性污染物，包括脂蛋白。而脂蛋白一般不存在于胆汁中，常常是专用的和申请专利的聚合物的成本使隔离比较昂贵。该聚合物也有时与下游应用如质谱不相容，但是当下游分析与聚合物（RNA或蛋白质隔离）兼容，该方法是简单，快速，并且不需要特殊的设备。 尺寸排阻色谱法具有长运行时间下行的专用设备的需求，但允许大和小颗粒的精确分离。免疫亲和纯化产生最高purit外来体囊泡和甚至ÿ允许特定外来体的级分，如衍生上皮的隔离，但是这是以总产的成本。此外，它是有限的，需要一个第一富集步骤小样本体积，如果大量的要被处理并分离的外来体可能会由于结合的抗体失去功能活性或表现出改变的生物学功能。 考虑下游应用（S）和专门设备的可用性，当涉及到外来体的分离是很重要的。无论使用何种分离方法的分离/富集颗粒的表征是非常重要的。超速离心成立至今，一直被许多实验室所使用的"金标准"，由于外来体颗粒的稳健和快速（和符合成本效益，如果超速离心机可用）富集。

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。

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

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。

由于外来体太小，通过定期显微镜进行检测或流式细胞术，电子显微镜或纳米颗粒光学分析必须执行。所述纳米颗粒的光分析具有优于电子显微镜的是，也定量和提供尺寸分布和浓度。仪器介绍通过玻璃棱镜到样品精细聚焦的激光束。分离的囊泡的布朗运动经由EMCCD高灵敏度摄像机捕获和跟踪在帧接帧的基础。该纳米颗粒跟踪分析（NTA）软件措施这布朗运动逐帧计算的大小，模式和胆汁外来体制剂的分布。 图1示出了从人胆汁样品中分离外来体的一个典型NTA分析的结果。 可替代地，电子显微镜可以利用以确认分离的颗粒的尺寸，尽管没有定量。 图2A示出了用于外来体从人的胆汁中分离透射电镜的典型结果。 对所分离的颗粒的外来体性质进一步验证，Western印迹探测像TSG101或四旋CD63的蛋白质，在图2B所示的情况下，都必须使用。特定外来体蛋白不存在本身 ，而是外泌体中tetraspanins，尤其CD9，CD63，CD81和CD82与CD63和CD81富集称为经典外来体标记物，以及涉及在外来体形成像TSG101和阿利克斯21的蛋白质。像热休克蛋白的同源70（的Hsc70）和73（Hsc73）其它蛋白质，以及第二主要组织相容性复合体（MHC）类分子，也可发现富集在外来体与一些像Hsc73和外周膜相关prote在乳脂肪球-表皮生长因子-因子8（Mfge8）是用于从树突细胞21所分泌的外来体相当具体。 图3示出了各种从人体胆汁外来体中提取的miRNA种类的典型的实时扩增曲线。由于外来体，不像细胞，缺乏可靠的标准，如18S或28S rRNA的或管家基因像GAPDH和β肌动蛋白用于标准化，用合成的miRNA扣球是重要的。的合成的miRNA应该从线虫不同物种像CEL-的miR 39导出到使一个有效正常化的表达水平。 再现性是至关重要的胆汁是尽快和未加工的冷冻处理之前为这些条件导致存在于胆汁miRNA的降解。另一方面，一旦分离，exoso<html> MES是非常稳定的并且在RT贮存48小时或最多三个冻融引起在至少两个物种的miRNA的水平可忽略不计的影响相当抗性，虽然针对感兴趣的miRNA的稳定性，必须凭经验确定。 <img alt="图1" src="/files/ftp_upload/54036/54036fig1.jpg" /> 图1（ 纳米），NTA分析表征人类胆管胆汁的外来外来体在PBS以1:1的比例稀释:600。从人的胆汁中分离电动车（A）粒度分布和浓度。被确定的平均大小是该样品约97纳米（x轴:外来体大小，y轴:外来体浓度为每大小）。在A中的大小范围分析相同样品的（B）的样品快照示出囊泡范围从30-110纳米。="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54036/54036fig2.jpg" /> 图2.核查的大小人胆汁70-110纳米的准备透射电子显微镜和现在的球形结构的印迹。（A）透射电子显微镜（TEM） 的外来体的性质 。比例尺为100nm。 （B）胆道外来体的Western blot分析显示了典型的外来体标记蛋白TSG101和CD63存在。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54036/54036fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54036/54036fig3.jpg" /> 图3.从胆汁中分离的外来物种的miRNA的实时PCR。真实-时间一的miR阵列的PCR表明人类胆汁外来体（X轴，PCR循环数; Y轴，相对强度）提取多个品种的miR的放大。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54036/54036fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

isolation-profiling-microrna-containing-exosomes-from-human

Nanyang Technological University

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JoVE Journal

Medicine

10.2K 次观看.  Johns Hopkins University School of Medicine. 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.

视频: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

高密度脂蛋白的分离的非编码小RNA定量

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

科研

生物化学

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

用于向癌细胞输送 sirna 的外质体的制备

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

癌症研究

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

遗传学

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

免疫与感染

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

外来体的纯化和microRNA剖析来源于血液和文化传媒

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...