Glomax 20 20 detector

TargetScan v7.2 was used to predict the binding site between HMGB1 and miR-181c-5p. The relationship of miR-181c-5p and circPTK2 was analyzed using starBase v2.0. Dual luciferase assay was used to verify the target relationship between HMGB1 and miR-181c-5p, or miR-181c-5p and circPTK2. The wild-type (HMGB1-WT) or mutant sequences (HMGB1-MUT) of HMGB1 or circPTK2 were inserted into the pmirGLO vector (E1330, Promega, USA) to synthesize reporter plasmids. The reporter plasmids and miR-181c-5p mimic (M) or mimic control (MC) were transfected into BV2 microglia as needed. The dual luciferase system (D0010-100T, Solarbio, China) was used to measure luciferase activity. The luciferase activity of different groups was detected using a GloMax 20/20 detector (Promega, USA).

The wild-type or mutant sequences of LINC00174 or E2F7 were integrated into the pmirGLO vector (E1330, Promega, USA). Different recombinant plasmids together with miR-3127-5p (M) or mimic control (Blank) were transfected into SW480 or LOVO cells, respectively. Luciferase activities were measured in dual luciferase system (D0010-100T, Solarbio, China) by GloMax 20/20 detector (Promega). Imprint RNA Immunoprecipitation Kit (RIP, Sigma-Aldrich) was constructed to RNA immunoprecipitation (RIP) assays. Briefly, CRC cells were lysed using RIR lysisi buffer, and protein A/G beads conjugated with anti-Argonaute2 (Ago 2) was added to cell extract for 6 h at 4°C. After that, samples were incubated with roteinase K to isolate RNA-protein complexes. IgG was served as control. Finally, immunoprecipitated RNA was subjected to RT-qPCR detection.