Fragment of predicated wild-type binding site (WT) or mutated sequences (MUT) was inserted in PmirGLO Firefly luciferase reporter (Promega, USA). Afterwards, NSCLC cells were co-transfected with reporter vector or Renilla luciferase control plasmid and miRNA mimic or miR-NC (negative control). At 48 h post-transfection, relative Firefly/Renilla luciferase activities were determined using a One-Step Firefly Luciferase Assay Kit (Biovision, USA).
Pmirglo firefly luciferase reporter
Manufactured by Promega
Sourced in United States
The PmirGLO Firefly luciferase reporter is a plasmid-based reporter system that utilizes the firefly luciferase gene as a reporter for gene expression analysis. The system provides a bioluminescent readout that can be used to quantify the activity of regulatory sequences, such as promoters and enhancers, in various cell types.
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2 protocols using pmirglo firefly luciferase reporter
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Luciferase Assay for miRNA Binding
2
Luciferase Assay for miRNA Binding
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To demonstrate the functional interaction, the sequence containing the wild-type binding sites or the sequence with mutated-binding sites were cloned into the PmirGLO firefly luciferase reporter (Promega, E1330). The reporter and Renilla luciferase (hRlucneo) control plasmid were co-transfected into cells in the presence of miR-29c-3p mimic or miR-NC in a 24-well plate (1 × 10^5 cells/well) using Lipofectamine 3000 reagent. 48 h after the transfection, the PierceTM Renilla-Firefly Luciferase Dual Assay Kit was used to determine the luciferase activities (Thermo Fischer Scientific, USA). The firefly luciferase activity in each sample was normalized to the activity of the control Renilla luciferase [17 (link)].
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