Dual luciferase analysis system kit

The 293T cells were seeded in 96-well plates for 24 hours and were transfected with pGL3-SRF-luciferase reporter (GENEWIZ), luciferase control reporter vector, and pcDNA3.1 TCF7L1 plasmids in the presence of pcDNA3.1 FLAG vector. Transfection was performed with Lipofectamine 2000. At 24 hours after transfection, luciferase activity was measured using a dual luciferase analysis system kit (Promega).

The binding site of miR-425 to CPEB1 3′-untranslated regions (3′-UTR) was predicted in the bioinformatics software (http://www.targetscan.org). Luciferase reporter gene assay was performed using the dual luciferase analysis system kit (Promega). The wild type (WT) and mutant (MUT) pmirGLO-CPEB1-3′UTR vector (Guangzhou Yingxin Co., Ltd., Guangzhou, China) contained the miRNA binding sites. Lung cancer cells were co-transfected with the vector with miRNA mimic. After 48 h, passive lysis buffer sample (20 μL) and Luciferase Assay Reagent II (100 μL) were added to cells and absorbance was measured at 580 nm on a microplate reader. Also, Stop&Glo reagent (100 μL) was added into cells and absorbance was read at 460 nm [17 (link)].