A549 cells (1×105) were plated in a 24-well plate before transfection. Next day, cells were co-transfected with the control pcDNA3.1 empty vector (EV, 225 ng) or the wild-type TTF-1 plasmid (225ng)-based firefly luciferase reporter plus the renilla luciferase control vector pGL4.73 (50 ng, Promega) using the K-2 transfection reagent (Biontex, #T060). After transfection (48 hr later), cell lysates were prepared and firefly/renilla luciferase values were quantified using Firely & Renilla Dual Luciferase assay (Biotium, #30005-2) on a GloMax-96 plate reader (Promega). Firefly luciferase values were normalized to Renilla luciferase values and expressed as relative values. See Supplementary Information for additional experimental methods on RNA profiling, cholesterol efflux, intracellular cholesterol quantitation and animal studies.
Firely renilla dual luciferase assay
Manufactured by Biotium
The Firely & Renilla Dual Luciferase assay is a luminescence-based reporter system that measures the activities of two different luciferase enzymes, Firefly luciferase and Renilla luciferase, within the same sample. The assay provides a way to normalize experimental results by using the Renilla luciferase as an internal control, allowing for the accurate quantification of gene expression levels.
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2 protocols using firely renilla dual luciferase assay
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Luciferase Assay for TTF-1 Transactivation
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Luciferase Assay for TTF-1 Transactivation
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A549 cells (1×105) were plated in a 24-well plate before transfection. Next day, cells were co-transfected with the control pcDNA3.1 empty vector (EV, 225 ng) or the wild-type TTF-1 plasmid (225ng)-based firefly luciferase reporter plus the renilla luciferase control vector pGL4.73 (50 ng, Promega) using the K-2 transfection reagent (Biontex, #T060). After transfection (48 hr later), cell lysates were prepared and firefly/renilla luciferase values were quantified using Firely & Renilla Dual Luciferase assay (Biotium, #30005-2) on a GloMax-96 plate reader (Promega). Firefly luciferase values were normalized to Renilla luciferase values and expressed as relative values. See Supplementary Information for additional experimental methods on RNA profiling, cholesterol efflux, intracellular cholesterol quantitation and animal studies.
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