**3.2. Ago2-immunoprecipitation and PCR analysis of novel miRNAs**

After bioinformatic analysis of the sequence data, we further validated novel miRNAs by using a combination of Ago2-immunoprecipitation **(13)** followed by PCR-based miRNA detection (**14**). Briefly, 50 ml Dynabeads protein G slurry (Invitrogen) was immobilized with

20 mg human P4 anti-mouse Ago2 monoclonal antibody (clone 2D4, Wako Pure Chemical Industries, Osaka, Japan). One hundred fifty micrograms of human tissue P4 were homogenized in 1.5 ml of a cell lysis solution (provided in miRNAs isolation kit, Wako) using a Polytron PT1200C homogenizer (Kinematica AG, Lucerne, Switzerland) for 10 s at 4 8C, and then 1.5 ml of the cell lysis solution was added into the homogenized solution. Following incubation for 15 min on ice, lysate was centrifuged at 20 000 g for 20 min at 4 8C and filtered through a 0.8 mmSupor Acrodisc syringe filter (Pall Corporation, Ann Arbor, MI, USA). One milliliter of the filtered lysate was incubated with 25 ml of the anti-Ago2- Dynabead protein G for incubation for 60 min at 4 8C. After immunoprecipitation, Ago2associated RNAs were isolated from the immunoprecipitate according to the manufacture's protocol (Wako). We confirmed that the immunoprecipitate contained human P5 Ago2 protein of w100 kDa in size by western blot (data not shown). Non-immune human IgG (Sigma) was used as a control for Ago2-immunoprecipitation. Preparation of the cDNA library using the Ago2-associated RNAs and semi-quantitative PCR analysis of the abovementioned novel miRNA candidates were performed, as reported previously **(14)**. A small RNA-specific primer and a universal reverse primer RTQ-UNIr **(14)**, were used for amplification of each of the small RNAs. The PCR products were analyzed on a 12% polyacrylamide gel. The primers for the human GAPDH were used for negative control.
