Université de Lorraine, CNRS, INSERM, IBSLOR, F-54000 Nancy, France.
Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France.
Methods Mol Biol. 2021;2300:165-182. doi: 10.1007/978-1-0716-1386-3_15.
Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. Human plasma, a readily available sample for biomedical analysis, was reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin, and composition. Unbiased analysis of exRNA composition can be performed with prefractionation of plasma exRNA followed by library preparation, sequencing, and bioinformatics analysis. In addition to "mature," adaptor ligation-competent RNA species (5'-P/3'-OH), human plasma contains a substantial proportion of degraded RNA fragments, featuring 5'-OH/3'-P or cyclophosphate extremities, which can be made competent for ligation using appropriate treatment. Polyethylene glycol (PEG)-based precipitation kits for EV isolation yield a fraction that is highly contaminated by large RNPs and EV-associated RNAs. Purer EV preparations are obtained by using Proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC).
细胞外 RNA（exRNA）几乎由所有细胞类型分泌，目前已知其具有多种生理功能。人类血浆是一种易于用于生物医学分析的样本，据报道其中含有各种 exRNA 亚群，其中一些可能是血浆核糖核蛋白（RNP）的组成部分，而另一些则被包裹在不同大小、来源和组成的细胞外囊泡（EV）中。通过对血浆 exRNA 进行预分级，然后进行文库制备、测序和生物信息学分析，可以对 exRNA 组成进行无偏分析。除了具有衔接子连接能力的“成熟”RNA 物种（5'-P/3'-OH）外，人血浆中还含有大量降解的 RNA 片段，具有 5'-OH/3'-P 或环磷酸酯末端，可通过适当的处理使其具有连接能力。基于聚乙二醇（PEG）的 EV 分离沉淀试剂盒会产生一种高度污染大 RNP 和 EV 相关 RNA 的级分。通过蛋白酶 K 和 RNase A 处理以及分子筛层析（SEC）可以获得更纯净的 EV 制剂。
