RNA immunoprecipitation assay

We performed a RNA immunoprecipitation (RIP) assay using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Merck-Millipore; Bedford, MA, USA). Briefly, we harvested 80% confluent NSCLC cells and lysed them using the RIP lysis buffer. Next, we performed Argonaute 2 (Ago2) immunoprecipitation by incubating the cell lysate with magnetic beads conjugated with anti-Ago2 antibody or normal immunoglobulin G (IgG) (control; Merck Millipore). Ago2 is an important element of RNA-induced silencing complex, and it can promote the degradation of target mRNAs via its catalytic activity in gene silencing processes induced by miRNAs. After overnight incubation at 4°C, we extracted the immunoprecipitated RNA and evaluated the abundance of lncRNA PRRT3-AS1, miR-507, and HOXB5 mRNA using qRT-PCR.