Abstract
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.
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