RIP-chip, that is, immunoprecipitation of RNA-induced silencing complexes (RISC) with an Ago2-specific monoclonal antibody followed by RNA extraction and subsequent quantification of mRNAs on microarrays, has recently been utilized to identify mRNAs that are associated with the RNA-silencing machinery and are therefore targets of cellular miRNAs [35 (link)–38 (link)]. In brief, 200 μg of total muscle protein was diluted with 200 μL of PBS buffer (pH 7.4). For each sample, 25 μL of protein A/G plus agarose (Santa Cruz) was washed with PBS and incubated with 2 μg of rabbit anti-Ago2 (Abcam, MA, USA) or rabbit normal IgG (Santa Cruz) antibodies for 2 h at 4°C. The beads containing the immobilized anti-Ago2 antibody were then added to 400 μL of diluted serum and incubated for 4 h at 4°C. The beads were washed 3 times with 1% NT-2 buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 2 mM EDTA) and the mixture was split in half. One-half of each sample was eluted in 2 × SDS sample buffer and subjected to SDS/PAGE and immunoblotting with a mouse anti-Ago2 antibody (Santa Cruz, CA, USA) to detect Ago2. The other half of each sample was eluted in 600 μL of lysis/binding buffer from the mirVana miRNA Isolation Kit (Life Technologies, NY, USA) and processed for RNA isolation. The RNA pellet was used for oligo-dT purification and library generation.
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