The wild type (WT) and mutated (MUT) sequence of SIRT1 promoter region was fragmented from distal end to proximal end and constructed with firefly and Renilla luciferase cDNA into GV354 vector (GeneChem, Shanghai, China), respectively. HEK-293T cells were seeded into 24-well plates at the density of 5 × 104 cells/per well 24 h before transfection, and extracted constructs were then co-transduced with oe-NC/oe-FOXQ1 plasmids into cells, followed by12-h incubation at 37°C. Dual-luciferase reporter analysis was performed according to the manufacturer’s instructions (Promega, Madison, Wisconsin) and the luciferase activity of firefly and Renilla luciferases was evaluated using the dual luciferase reporter assay system (Promega, Madison, Wisconsin). The ratio of firefly luciferase activity to Renilla luciferase activity indicated the relative activity of luciferase.
Dual luciferase reporter analysis
Manufactured by Promega
The Dual-Luciferase Reporter Assay System is a laboratory tool that allows for the measurement and analysis of gene expression. It utilizes two different luciferase reporter enzymes to provide a quantitative report of gene activity.
Automatically generated - may contain errors
2 protocols using dual luciferase reporter analysis
1
SIRT1 Promoter Regulation Assay
2
Validating miR-17-5p Regulation of TLR4
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The TargetScan tool (version 7.2) was used to assess the putative binding sites between miR-17-5p and TLR4 3'UTR. Wild-type (WT) fragments of TLR4 3'UTR were ampli ed from genomic DNA and subcloned into a pmirGLO reporter vector (Promega, Madison, USA). This construct was termed TLR4 3'UTR-WT. TLR4 3'UTR mutations were performed through site-directed mutagenesis kit (Stratagene, San Diego, USA). The construct was termed TLR4 3'UTR-MUT. For luciferase activity, HEK293 cells were transfected using Lipofectamine 3000 reagent (Invitrogen) with miRNAs (miR mimic or NC mimic) and luciferase reporter vectors (TLR4 3'UTR-WT or TLR4 3'UTR-MUT). Forty-eight hours post-transfection, luciferase activity was assessed using dual luciferase reporter analysis (Promega).
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