The study of extracellular miRNA is one of the most dynamic areas of modern biomedical research. Oftentimes, there is a need to isolate miRNAs associated with a particular carrier, for example, a ribonucleoprotein complex. The most thoroughly investigated protein component of these complexes is Ago2. Complexes are commonly isolated by immunoprecipitation with specific antibodies. Here we compare three methods for immunoprecipitating Ago2/microRNA complexes from blood plasma. In the first protocol, anti-Ago2-antibodies are added to the plasma following protein A-sepharose. In second protocol, anti-Ago2-antibodies are bound to sepharose from the very beginning, and then mixed with plasma. The third protocol differs from the second in that sepharose is blocked by non-specific antibodies at the final stage. To compare the efficiency of these protocols, the levels of miR-16-5p, miR-21-5p, and miR-144-3p were analyzed after precipitation with anti-Ago2 antibodies and control antibodies. For miR-16-5p all protocols were efficient, for miR-21-5p only the second technique yielded results, while for miR-144-3p none of the protocols resulted in extraction. Thus, we conclude that different protocols for immunoprecipitation of microRNA/Ago2 complexes favor different miRNAs.

中文翻译：

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人体血浆中microRNA / Ago2复合物的免疫沉淀效率取决于协议

细胞外miRNA的研究是现代生物医学研究中最活跃的领域之一。通常，需要分离与特定载体例如核糖核蛋白复合物相关的miRNA。这些复合物中最深入研究的蛋白质成分是Ago2。通常用特异性抗体通过免疫沉淀分离复合物。在这里，我们比较了三种从血浆中免疫沉淀Ago2 / microRNA复合物的方法。在第一个方案中，将抗Ago2-抗体添加到蛋白A-琼脂糖之后的血浆中。在第二种方案中，抗Ago2抗体从一开始就与琼脂糖结合，然后与血浆混合。第三种方案与第二种方案的不同之处在于，琼脂糖酶在最后阶段被非特异性抗体封闭。为了比较这些协议的效率，用抗Ago2抗体和对照抗体沉淀后分析miR-16-5p，miR-21-5p和miR-144-3p的水平。对于miR-16-5p，所有协议都是有效的，对于miR-21-5p，只有第二种技术才可产生结果，而对于miR-144-3p，没有协议可导致提取。因此，我们得出结论，用于microRNA / Ago2复合物免疫沉淀的不同方案偏爱不同的miRNA。