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.

中文翻译：

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一种用于外泌体和无细胞miRNA分离和定量的新方法。

在外泌体中使用疾病特异性的microRNA（miRNA）签名对于作为生物标志物或直接治疗靶标的临床应用已大有希望。然而，由于商业技术在数量和质量上都有缺点，因此迫切需要一种用于miRNA外来体富集和定量的新方法，以用于其临床应用。为了克服这些缺陷，我们开发了一种纯化外泌体的新方法，随后提取了miRNA，随后进行了定量逆转录聚合酶链反应（RT-qPCR），并将我们的检测方法与商业技术进行了比较。为了建立这些方法，检查了许多试剂，参数及其组合。我们用于外来体提取的新技术基于避免了共沉淀血浆蛋白的甘露糖醛酸-古洛糖酸酯聚合物（MGP）。通过蛋白质印迹，纳米颗粒跟踪分析（NTA）和共聚焦显微镜检查分离出的外泌体的质量，浓度和生物学活性。离液盐和非离液盐的组合用于从血浆，血清和外泌体中提取miRNA，从而排除了有害成分，例如苯酚/氯仿。通过RT-qPCR验证了miRNA提取的性能。化学和TaqMan探针也针对RT-qPCR进行了优化。在合成miR-16和miR-142的系列稀释液中测试了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，