CMVECs were seeded into 96-well plates in triplicate at a density of 5 × 103 cells per well 24 h before transfection. The circHIPK3 sequences containing wild-type miR-29a binding sites were synthesized and inserted into the pmirGLO luciferase vector (GeneCreate, Wuhan, China), and an empty vector was used as a control. The cells were cotransfected with a mixture of firefly luciferase reporter vector, pRL-TK vector (Renilla luciferase control reporter vector) (Promega), and miR-29a mimics or negative control by using Lipofectamine 2000. In addition, 200 ng of pmirGLO-IGF-1-WT or pmirGLO- IGF-1-Mut reporter plasmids was cotransfected into cells with 50 nM miR-29a mimics using Lipofectamine 2000. Cells were harvested 48 h after transfection, and luciferase reporter assays were performed using a dual luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions. Relative luciferase activity was normalized to the Renilla luciferase internal control.
Prl tk vector renilla luciferase control reporter vector
Manufactured by Promega
The PRL-TK vector is a Renilla luciferase control reporter vector. It expresses the Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase (HSV-TK) promoter.
Automatically generated - may contain errors
2 protocols using prl tk vector renilla luciferase control reporter vector
1
Luciferase Assay for circHIPK3-miR-29a Interaction
2
3′-UTR Luciferase Assay for miR-508-5p
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Wild-type (Wt) and mutant (Mut) 3′-UTR of LOC102724163 and MUC19 were cloned downstream of the firefly luciferase gene in the pGL3 vector (Promega Corporation). 293T cells were plated in 96-well plates at a density of 5×103 cells/well with DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin in a cell incubator at a constant temperature of 37°C with 5% CO2, 24 h prior to transfection. Cells were co-transfected with the firefly luciferase reporter vector, the pRL-TK vector (Renilla luciferase control reporter vector; Promega Corporation) and miR-508-5p mimic. After 48 h of transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega Corporation).
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