Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.
Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.
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DOI：
10.1371/journal.pone.0136133
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Noerholm M
中科院分区：
文献类型：
--
作者：
Enderle D;Spiel A;Coticchia CM;Berghoff E;Mueller R;Schlumpberger M;Sprenger-Haussels M;Shaffer JM;Lader E;Skog J;Noerholm M
外来体和其他细胞外囊泡（通常称为EV）由于其在诊断和治疗中的潜在应用而引起了很多关注。这些囊泡的内容物是深入研究的主题，并且EV内部RNA的相对较新的发现引起了人们对这些RNA的生物学功能以及它们作为癌症和其他疾病的生物标志物的潜力的兴趣。传统的基于超离心分离的EV分离方案是劳动密集型的，并且具有显著的可变性。开发具有稳健、可再现性能的方法的各种尝试尚未完全成功。在这里，我们报告了一种基于旋转柱的方法的开发和表征，用于从血清和血浆中的EV中分离总RNA。与超离心相比，该方法分离出相等或更高量的高纯度RNA，对囊泡RNA的特异性高于非囊泡RNA。旋转柱能够处理高达4 mL的样品体积，从而能够检测血清和血浆中的低丰度转录物。我们的结论是，该方法是对传统方法的改进，提供了一种更快，更标准化的方法，以实现可靠的高质量的RNA制剂从EV的生物流体，如血清和血浆。Qiagen最近推出了第一个利用这种新方法的试剂盒，名称为“exoRNeasy血清/血浆Maxi试剂盒”。
Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as “exoRNeasy Serum/Plasma Maxi Kit”.