Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
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DOI：
10.3402/jev.v5.31655
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发表时间：
2016
影响因子：
16
通讯作者：
Yáñez-Mó M
中科院分区：
文献类型：
--
作者：
Andreu Z;Rivas E;Sanguino-Pascual A;Lamana A;Marazuela M;González-Alvaro I;Sánchez-Madrid F;de la Fuente H;Yáñez-Mó M
细胞外小泡(EVS)是一种有效的非侵入性生物标志物。然而，目前的方法是耗时的，很难转化为临床实践。为了分析EV包裹的循环miRNA，我们寻找了一种快速、简便、经济的方法来富集人血清标本中的EV。我们比较了几种协议和商业试剂盒分离电动汽车的效率。对基于沉淀、柱子或过滤系统的不同方法进行了测试，并与最经典的肠道病毒分离方法超速离心法进行了比较。EV样本的纯度和数量通过纳米颗粒跟踪分析和针对主要EV蛋白标志物的蛋白质印迹或细胞术进行评估。为了验证生物标记物，测定了EV组分中一组miRNAs的水平，并与它们在总血清中的水平进行了比较。用沉淀法分离的EVS富含与包裹在EVS(miR-126、miR-30c和miR-143)中的miRNAs对应的miRNAs亚群，而miR-21、miR-16-5p和miR-19a的检测率与总血清相比非常低。我们的结果表明，使用聚乙二醇法(聚乙二醇法)沉淀是一种简单而廉价的血清EVS浓缩方法，用于miRNA分析。在蛋白质产量和miRNA产量方面，聚乙二醇的总体性能与其他商业沉淀剂非常相似或更好，但与它们相比，聚乙二醇要便宜得多。其他方法的结果较差，主要是在通过qPCR分析评估miRNA时。在对人体样本进行的纵向研究中，我们使用聚乙二醇沉淀法，证明可以在冷冻样本中评估miRNA，最长可储存8年。我们报告了一种基于折叠EV检测相对于血清的平均值的截止值的方法，该方法提供了对给定miRNA的包裹度的估计。
Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation-based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR-126, miR-30c and miR-143), while the detection of miR-21, miR-16-5p and miR-19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut-off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.