Imprint rna immunoprecipitation rip kit

Through an Imprint RNA Immunoprecipitation (RIP) Kit (Sigma-Aldrich), RIP assay was conducted. The hypoxia-induced PC12 cells were harvested and then lysed in RIP lysis buffer (Invitrogen). After being added to magnetic beads (Millipore), the whole cell lysate was then incubated with negative control anti-IgG (Abcam) or anti-Ago2 (Abcam). The immunoprecipitated RNA was subjected to qRT-PCR analysis after the isolation and purification processes.

The Imprint^® RNA Immunoprecipitation RIP Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to analyze the interaction between hsa_circ_11780 and miR-544a according to the manufacturer’s instruction. Briefly, H226 and A549 cells transfected with miR-544a mimics or miR-NC were cultured 48 h before anti-AGO2 RIP assay. After the tumor cells were lysed by the lysis buffer (RNase inhibitor and protease inhibitor), the suspension was incubated with magnetic beads (conjugated with Ago2 antibody) (Abcam, San Francisco, CA, USA), or negative control IgG. Finally, the antibody binding RNA was identified by RIP-qPCR assay using the respective target primers.

RIP assay was performed to explore whether RNA induced silencing complex (RISC) contained NEAT1 and miR-34a-5p in 5-8F cells using an Imprint RNA Immunoprecipitation (RIP) kit (Millipore, Billerica, MA, USA) referring to the directions of the manufacturers. In brief, 5-8F cells transfected with miR-34a-5p mimics were lysed in RIP lysis buffer (Takara, Beijing, China) and were incubated with protein A/G magnetic beads and Argonaute-2 antibody (anti-Ago2, Abcam, Cambridge, UK) or IgG antibody (anti-IgG, Abcam). Then, cell lysates in the miR-34a-5p mimics group were centrifuged. The supernatants were marked as output, and immunoprecipitation complexes were named as IP. Western blot analysis was performed to validate successful RIP with anti-Ago2. Lastly, qRT-PCR assay was used to detect NEAT1 enrichment in the RIP RNA fraction.