Institute of Carcinogenesis, Blokhin National Medical Research Center of Oncology, Moscow, 115478, Russia.
Faculty of Biology, Lomonosov Moscow State University, Moscow, 111234, Russia.
Biochemistry (Mosc). 2022 Nov;87(11):1354-1366. doi: 10.1134/S0006297922110141.
Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.
细胞外囊泡（EVs）包含活性生物分子，包括 miRNA，其组成反映了细胞在病理过程中发生的表观遗传变化，特别是恶性转化。关于 EV 在致癌作用中的作用的累积数据刺激了对 EV 衍生的癌症标志物的研究。限制这一科学方向发展的最重要因素是缺乏 EV 从生物体液中分离的方法和分析其分子含量（包括 miRNA 组成）的“金标准”。在这里，我们首先研究了从小鼠腹水来源的 EV 中分离小 RNA 的各种方法对后续 miRNA 分析的效果。不同商业试剂盒的比较显示，基于酚氯仿提取的方法具有优势：Total Exosome RNA & Protein Isolation Kit 和 miRNeasy Serum/Plasma Kit。对小 RNA 转录组的分析表明，EV 中存在各种分子，其中 miRNA 的比例平均为 6%，使用 Total Exosome RNA & Protein Isolation Kit 可达到 10%。PureLink miRNA Isolation Kit 的效率最低。miRNeasy Advanced Serum/Plasma Kit 显示出小 RNA 馏分的最高浓度，但 miRNA 的比例并未超过 miRNeasy Serum/Plasma Kit 和 Total Exosome RNA & Protein Isolation Kit 的比例。此外，对单个分子的 RT-PCR 分析表明，使用 miRNeasy Advanced Serum/Plasma Kit 时，每个研究 miRNA（miR-1246、miR-200b-5p、miR-200c-3p 和 miR-23a-3p）的水平都较低。总之，根据所研究的特性（包括小 RNA 浓度、根据 bioanalyzer 和测序结果的 miRNA 百分比以及通过 RT-PCR 检测到的个别 miRNA 水平），Total Exosome RNA & Protein Isolation Kit 和 miRNeasy Serum/Plasma Kit 可被认为是性能最佳的试剂盒。
