Research and Development, AJ Innuscreen GmbH, Berlin, Germany.
Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
RNA Biol. 2020 Apr;17(4):425-440. doi: 10.1080/15476286.2020.1721204. Epub 2020 Feb 10.
The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.
利用外泌体中 microRNAs（miRNAs）的疾病特异性特征已成为临床应用的一种有前途的方法，无论是作为生物标志物还是直接治疗靶点。然而，由于商业技术在数量和质量上存在缺陷，迫切需要一种新的外泌体富集和 miRNA 定量方法用于其临床应用。为了克服这些缺陷，我们开发了一种新的外泌体纯化方法，随后进行 miRNA 提取，然后进行定量逆转录聚合酶链反应（RT-qPCR），并将我们的测定方法与商业技术进行了比较。为了建立这些方法，我们研究了许多试剂、参数及其组合。我们的外泌体提取新技术基于甘露糖醛酸-葡萄糖醛酸聚合物（MGP），可避免共沉淀血浆蛋白。通过 Western blot、纳米颗粒跟踪分析（NTA）和共聚焦显微镜检查了分离的外泌体的质量、浓度和生物活性。采用一种包含离子变性剂和非离子变性剂的盐混合物从血浆、血清和外泌体中提取 miRNA，从而可以排除诸如酚/氯仿等有害成分。通过 RT-qPCR 验证了 miRNA 提取的性能。还对 RT-qPCR 的化学和 TaqMan 探针进行了优化。通过对合成的 miR-16 和 miR-142 的系列稀释液测试了 RT-qPCR 的灵敏度、效率和线性。我们建立的程序涵盖了 miRNA 分析的所有步骤，并可高性能地测量血浆、血清和其他体液中游离和外泌体 miRNA 的水平。
