VSMCs and 293T cells were seeded on 6-well plates at a density of 6×105 cells/cm2 and cultured overnight in a humidified atmosphere with 5% CO2 at 37°C. Cells were then transfected with 20 nM miR-17 mimic or 20 nM inhibitor or 20 nM control miRNA (Guangzhou RiboBio Co., Ltd., Guangzhou, China) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 24 h, cells were collected for western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses.
To construct the pGL3-RB-3′ untranslated region (3′UTR), the full length 3′UTR of the human RB mRNA was cloned into the pGL3-control vector (Promega Corp., Madison, WI, USA). To construct the miR-17 promoter reporter genes, the DNA fragment upstream of the transcription start site of the miR-17 gene was inserted into the pGL3-basic vector (Promega Corp.). For luciferase reporter assays, 293T cells were transiently transfected with miR-17 mimics and Renilla-luciferase reporter plasmids (Promega Corp.) containing the pGL3-RB-3′UTR (with the binding region: AGGCGCUU) or pGL3-RB-3′UTR MUT (with the mutant binding region: UUCGUGAA) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were collected and reporter gene activity was determined using the dual-luciferase assay system (Invitrogen; Thermo Fisher Scientific, Inc.). Renilla luciferase activity was used for normalization of transfection efficiency. Reporter luciferase activity assay was also used to assess the function of NF-κB p65 regulating miR-17 promoter activity. There were three NF-κB p65 binding sites in the regulatory sequence of the miR-17 gene. Full paper
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