Dual-Luciferase® Reporter Assay System

Protocol tips

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	Use 30 ng of Renilla luciferase vector, pGL4.74 vectors to transfect cells. Transfect for 48h and harvest cells.

Protocol tips
Use 30 ng of Renilla luciferase vector, pGL4.74 vectors to transfect cells. Transfect for 48h and harvest cells.

Publication protocol

The miR-542-3p candidate targeting sequence of IGFBP1 gene (5′-GAUGAAAUAAUGUUCUGUCACG-3′, part of the NCBI RefSeq ID of NM_000596) and its mutated sequence (5′-GAUGAAAUAAUGUUUCUGUGAG-3′) were inserted downstream of the pGL4.13 luciferase expression vector (Promega Corporation, Madison, WI, USA). The resulting constructs were named as IGFBP1-wt and IGFBP1-mut, respectively. Those two vectors with an amount of 200 ng and a final concentration of 30 nM miR-542-3p mimic were co-transfected to HESCS treated with 0.5 mM 8-br-cAMP and 10−6 M MPA for 6 days before transfection. As a negative control, native pGL4.13 luciferase vector was also transfected in parallel. For normalisation, 30 ng of Renilla luciferase vector, pGL4.74 (Promega), was also transfected into all samples. Transfections with all vectors and RNA combinations were repeated three times. After 48 h, the cells were harvested with PLB reagent (Promega), following which the firefly and Renilla luciferase activity was measured in each well using the dual luciferase assay kit (Promega) with the ARVO MX plate reader. The relative firefly luciferase activity was calculated by normalisation to the Renilla luciferase activity.

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Manufacturer protocol

Download the product protocol from Promega for Dual-Luciferase® Reporter Assay System below.

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