Experimental Identification of MicroRNA Targets by Immunoprecipitation of Argonaute Protein Complexes
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MicroRNAs (miRNAs) represent a class of small noncoding RNAs that negatively regulate gene expression. Intensive research during the past decade has established miRNAs as key regulators of many cellular pathways. MiRNAs have also been implicated in a number of diseases including various forms of cancer. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and function in multi-protein complexes. MiRNAs guide Ago protein complexes to partially complementary sequences typically located in the 3′ untranslated region (UTR) of their target mRNAs leading to the inhibition of its translation and/or its destabilization. To understand the biological roles of miRNAs, it is essential to identify the mRNA targets that they regulate. Because of the low degree of complementarity between the miRNA and its target sequence, it is often difficult to find targets computationally. Therefore, biochemical methods are needed to identify miRNA targets experimentally. The availability of highly specific monoclonal antibodies against Argonaute proteins allows for the isolation of functional Ago-miRNA–mRNA complexes from different cell lines, tissues, or even patient samples. Here we provide a detailed protocol for isolation and identification of miRNA target mRNAs from immunoprecipitated human Ago protein complexes.
Key wordsArgonaute proteins MicroRNAs Immunoprecipitation Gene silencing mRNAs Dicer Drosha
We thank Sabine Rottmüller and Bernd Haas for technical assistance, Elisabeth Kremmer for antibody production, and Vladimir Benes for affymetrix array analysis. This work was in part supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, FO855), the European Union (LSHG-CT-2006-037900), and the Max-Planck-Society.
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