Comparison of serum exosome isolation methods on co-precipitated free microRNAs
Comparison of serum exosome isolation methods on co-precipitated free microRNAs
复制标题
共沉淀游离 microRNA 的血清外泌体分离方法的比较。
DOI：
10.7717/peerj.9434
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发表时间：
2020-08-28
期刊：
影响因子：
2.7
通讯作者：
Xia, Weiliang
中科院分区：
文献类型：
--
作者：
Cheng, Yirui;Qu, Xiangyun;Xia, Weiliang
背景。外泌体是纳米级的细胞外囊泡，含有不同的生物分子，如蛋白质和介导细胞间通讯的microrna （mirna）。近年来，许多研究报道了外泌体mirna在疾病发展中的重要功能以及作为诊断生物标志物的潜在临床应用。目前，提取外泌体最常用的方法是超离心（UC）和基于沉淀的商业试剂盒（如ExoQuick）。一般来说，UC和ExoQuick方法都可以将污染蛋白与外泌体一起共分离，UC方法产生的外泌体纯度甚至高于ExoQuick。然而，这两种方法在共沉淀游离mirna上的比较尚不清楚。在这项研究中，我们用UC和ExoQuick从外源性添加了细胞- mir -39的人血清中分离出外泌体，并比较了两种方法提取的外泌体中细胞- mir -39共沉淀的比例。使用外源性细胞- mir -39作为血清中的游离mirna，我们得出结论，ExoQuick共同分离了一小部分游离mirna，而UC几乎没有沉淀任何游离mirna。我们还发现，在37℃下孵育1小时可以降低游离mirna的比例，并且当使用RNase A时，miR-126和miR-152等外泌体mirna也减少。总之，我们的研究结果为进一步研究外泌体mirna提供了关于血清外泌体分离方法细节的重要信息。
Background. Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.Methods. In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods.Results. Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 degrees C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.