Transcriptional activity assays were performed using the Luciferase Assay System (Genepharma, China) according to the manufacturer’s instructions37 (link). Briefly, cells were transfected with siRNAs targeting β-catenin or the scramble control. Cells were co-transfected with the M50 Super 8× TOP Flash or M51 Super 8× TOP Flash by Lipofectamine 3000 after 24 h post transfection. In some of the experiments, cells were also co-transfected with TMEM170B expression vector and miR-27a mimic or inhibitor. The luciferase activity of each sample was normalized against Renilla luciferase activity to monitor transfection efficiency. Twenty-four hours after transfection of plasmids, cells were lysed and luciferase activity was measured using the dual-luciferase reporter (DLR) assay Kit (Promega). Firefly and Renilla luciferase activities were determined using a luminometer and normalized.
Luciferase assay system
Manufactured by GenePharma
Sourced in China
The Luciferase Assay System is a laboratory equipment designed for the detection and quantification of luciferase enzyme activity. The system utilizes bioluminescent reactions to measure the expression of luciferase reporter genes in cell-based assays. It provides a sensitive and rapid method for analyzing gene expression, cell signaling, and other biological processes.
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2 protocols using luciferase assay system
1
Wnt/β-catenin signaling reporter assay
2
Luciferase Assay for miR-29b Targets
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Luciferase Assay System (GenePharma Co., Ltd., Shanghai, China) was used to confirm the target relationship between miR-29b and the potential downstream target genes. Briefly, wild type 3′-UTR sequence of target genes containing miR-29b binding site and mutant 3′-UTR sequence of those target genes were inserted into Pmir-GLO luciferase reporter vectors (GenePharma Co., Ltd., Shanghai, China). These Pmir-GLO vectors were co-transfected with miR-29b mimic/inhibitor or mimic/inhibitor control (GenePharma Co., Ltd., Shanghai, China) into human bronchial epithelial cells (16HBE cells). The cells were harvested and relative luciferase activity (activity firefly/activity renilla) was detected.
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