Wide type (WT)-MEG3 containing binding sites were amplified by PCR using Ultra HiFidelity PCR Kit (TIANGEN, Beijing, China), and mutation type (MUT)-MEG3 was synthesized by Sangon (Shanghai, China). The obtained MUT-MEG3 and WT-MEG3 sequences were cloned into pmirGLO vectors. The vectors carried with WT-MEG3 were co-transfected with miR-29 c mimics, miR-29 c inhibitors, or miR-NC into MRC-5 cells via lipofectamine 3000 ((Life Technologies, Carlsbad, USA). The co-transfection procedures of MUT-MEG3 were consistent with that of the WT-MEG3 group. Luciferase activities were surveyed 48 hours after transfection utilizing the Dual-Glo luciferase system kit (Promega, Madison, USA) [20 (link)].
Dual glo luciferase system kit
Manufactured by Promega
Sourced in United States
The Dual-Glo luciferase system kit is a reagent kit designed for the measurement of firefly and Renilla luciferase reporter gene activities in mammalian cell extracts. The kit provides the necessary reagents for the sequential detection of the two luciferase reporter enzymes.
Automatically generated - may contain errors
2 protocols using dual glo luciferase system kit
1
Investigating MEG3-miR-29c Regulatory Axis
2
FOXO3a 3'UTR Luciferase Assay
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The plasmids of the wild-type (WT) and mutated (MUT) 3′-untranslated region (UTR) of FOXO3a constructed into the pGL3-Luc vector (Promega Corporation) were purchased from Shanghai GeneChem Co., Ltd. For the luciferase assay, 2.0×104 A549 cells were cultured in a 48-well plate overnight and co-transfected with pGL3-WT FOXO3a 3′-UTR (100 nM) or pGL3-MUT FOXO3a 3′-UTR (100 nM) and miR-21-3p mimic (50 nM) or mimic NC (50 nM) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were transfected at 37°C for 48 h. At 48 h post-transfection at 37°C, relative luciferase activities were analyzed using a Dual-Glo luciferase system kit (Promega Corporation) according to the manufacturer's protocol with Renilla luciferase activity as the normalization control.
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