Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs
Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs
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DOI：
10.1080/20013078.2018.1555410
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发表时间：
2019-01-01
影响因子：
16
通讯作者：
Pitkanen, Asla
中科院分区：
文献类型：
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作者：
Karttunen, Jenni;Heiskanen, Mette;Pitkanen, Asla
血浆细胞外囊泡 (EV) 中含有的 microRNA (miRNA) 提供了相对较少探索的脑部疾病生物标志物来源，可以通过非侵入性方式获得。然而，从血浆中分离电动汽车的方法仍在开发中。对于 EV 分离，重要的是要确保去除无囊泡 miRNA，这些 miRNA 约占血浆 miRNA 的三分之二。基于膜颗粒沉淀的 EV 分离是一种颇具吸引力的方法，因为其操作简单且产量高。在这里，我们评估了基于沉淀的方法从少量大鼠血浆中获取富集的 EV 特异性 miRNA 的性能。我们对沉淀分离的 EV 颗粒和全血浆进行了尺寸排阻色谱 (SEC)。使用纳米颗粒跟踪分析 (NTA)、蛋白质和 miRNA 浓度测定以及四种 miRNA（miR-142-3p、miR-124-3p、miR-23a、miR-122）的液滴数字聚合酶链反应对 SEC 组分进行分析。还使用透射电子显微镜 (TEM) 分析了沉淀分离的 EV 和血浆中选定的 SEC 部分。基于沉淀的 EV 分离可共沉淀 9% 至 15% 的血浆蛋白和 21% 至 99% 的无囊泡 miRNA，具体取决于各个 miRNA。此外，主要存在于 EV 组分中的 miR-142-3p 的量在 EV 组分中减少，表明在基于沉淀的分离过程中部分丢失。蛋白质印迹和 TEM 显示沉淀分离的 EV 沉淀中存在蛋白质和脂蛋白污染。我们的研究结果表明，基于沉淀的方法不足以纯化血浆中含有 EV 的 miRNA 货物。 NTA 测得的颗粒数较高，但这主要是由于脂蛋白污染所致。尽管部分无囊泡 miRNA 被去除，但在基于沉淀的方法分离的血浆 EV 颗粒中，无囊泡 miRNA 仍然占主导地位。
The microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle-free miRNAs, which account for approximately two-thirds of plasma miRNAs. Membrane particle precipitation-based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation-based method to obtain enriched EV-specific miRNAs from a small volume of rat plasma. We performed size-exclusion chromatography (SEC) on precipitation-isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR-142-3p, miR-124-3p, miR-23a, miR-122). Precipitation-isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation-based EV isolation co-precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle-free miRNAs, depending on the individual miRNAs. In addition, the amount of miR-142-3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation-based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation-isolated EV-pellets. Our findings indicate that a precipitation-based method is not sufficient for purifying plasma EV-contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle-free miRNA is removed, vesicle-free miRNA still dominates in plasma EV pellets isolated by the precipitation-based method.