To measure HH or WNT signaling responses, A549 cells or MEFs were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) with GLI response-element reporter (8xGliBS-Luc) plasmid or TCF reporter plasmid (TOPflash), respectively, and pTK-Renilla-Luciferase (pRL-TK) plasmid. 24 h following transfection, the cells were treated with SHH CM or WNT CM and the indicated compounds for another 24 h. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System in a SpectraMax iD3 (Molecular Devices). The Firefly luciferase activities were normalized to the Renilla luciferase activities. Data are reported as the mean of triplicate measurements ± SD. Where indicated, A549 cells were also transfected with LRP6-pCS2 (a gift from Dr. Xi He, Addgene plasmid # 27,242), pCMV5-3xFLAG-DVL2 and pCS2-CSNK1E, or pEF-MYC-CTNNB1 (a gift from Dr. Henry Ho, University of California, Davis).
Dual luciferase reporter assay system
Manufactured by Molecular Devices
Sourced in United States
The Dual-Luciferase Reporter Assay System is a laboratory tool used to measure the activity of two different luciferase reporter enzymes within a single sample. The system provides a quantitative method for analyzing gene expression and regulatory elements.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
Os rc 2, by GenePharma (1 mentions)
Spectramax m5 multi mode microplate reader, by Molecular Devices (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Snapgene software, by GSL Biotech (1 mentions)
Pex 3 vector, by GenePharma (1 mentions)
Pmirglo dual luciferase vector, by GenePharma (1 mentions)
Multifunctional microplate reader, by Molecular Devices (1 mentions)
7 protocols using dual luciferase reporter assay system
1
Measuring Hedgehog and Wnt Signaling
2
Assessing ARE Activation After RT
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The ARE activation after RT was tested by assessing the luciferase activity. Firstly, U251-AREgv and U87-AREgv cells were plated at 2×103 cells/well in 96-well plates in 100 µL media and incubated overnight before RT in hypoxia. Luciferase activity was assessed by Dual-Luciferase® Reporter Assay System (Molecular Devices M3, CA, USA). Normalization of data was done by cell number.
3
Luciferase Reporter Assay for GALNT2
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The dual-luciferase vectors were acquired from Jiangsu Dongxuan Gene Technology Co. Ltd., and harbored the 3ʹ-UTR of GALNT2 with seed sequences. The corresponding mutant vectors were generated by ligating the 3-bp mutations with the aforementioned seed sequences. Next, the GALNT2/mutant vectors and miR-139-5p mimics were co-incorporated into Caki-1 and OS-RC-2 cell lines by Gene Pharma (Gene Pharma; Shanghai GenePharma Co. Ltd., CHINA). Luciferase activity quantification was performed 48 h later with the use of Dual-Luciferase Reporter Assay System (Molecule Device, Molecular Devices, LLC., U.S.A.).
4
Dual-Luciferase Assay for miRNA Targeting
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Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's instructions. Briefly, A549-miR-199a-5p-OE and A549-pGFP-vector cells were plated in 96-well plates at the density of 3.5×103 cells/well and allowed to attach overnight at 37°C. psiCHECK-2 reporter plasmids (200 ng) containing the wild-type sequence which was 5′-GACACTTCGG-3′ or mutated coding sequence which was 5′-GGGTGTGTCG-3′ were transfected into the cells at 90% confluence. The wild-type sequence was obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/gene/8878 ) and determined using Snapgene Software (GSL Biotech, LLC). Following 24-h transfection, the cells were lysed using the Passive Lysis Buffer from the Dual-Luciferase Reporter Assay system kit, and the luciferase activity was measured by a SpectraMax M5 Multi-Mode microplate reader (Molecular Devices, LLC). The firefly luciferase activity was normalized to that of Renilla luciferase, and the relative luciferase activity was calculated by dividing the luciferase activity value of the A549-miR-199a-5p-OE group by that obtained in vector group. The sequences used for plasmid sequencing were Rluc-Forward 5′-AGGACGCTCCAGATGAAATG-3′ and psiCHECK-2-Reverse, 5′-ACTCATTTAGATCCTCACAC-3′.
5
Luciferase Reporter Assay for HIF-1α
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When the cell density reached 1 × 105 cells/well, the cells were transfected with luciferase constructs either with or without HIF-1α plasmids. After transfection for 24 h, the medium was discarded. The cells were washed gently, and cell lysis buffer was added to the cells for 15 min. All of the cell lysate was collected into a tube and then centrifuged at 16,000 rpm for 5 min, after which the supernatant was collected for subsequent detection. A total of 20 μl of cell lysis supernatant was then added into a detection tube, followed by 100 μl of firefly luciferase reaction buffer containing a substrate balanced to room temperature, and the activity of luciferase was immediately detected using a Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Molecular Devices, USA). Then, 100 μl of Renilla substrate was added to detect the Renilla activity. A Luciferase Reporter Assay kit (MA0518; Dalian Meilun Biotechnology) was used to detect the luciferase activity.
6
Regulation of HIF-1α and PFKP by miR-186
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HIF-1α mRNA 3′UTR (untranslated regions) containing the predicted binding sites were selected. The theoretical binding sequence of miR-186 in HIF-1α gene and its mutant sequence was designed. The 3′UTR fragment of HIF-1α and its mutant were cloned into a firefly luciferase reporter construct pmirGLO Dual-luciferase vectors (GenePharma, Suzhou, China), respectively. HEK 293T cells were cotransfected with HIF-1α-3′UTR-Wt (or HIF-1α-3′UTR-Mut) and agomir (or agomir-186-NC). After transfection, cells were harvested, lysed and subjected for the dual luciferase reporter assay system (Molecular Devices).
By screening the promoter region of PFKP, a binding motif (CACGC) of HIF-1α was discovered. To determine the responsive HIF-1α-binding sites in the human PFKP promoter, promoter activities were measured using Dual-Luciferase Reporter Assay System. Human full-length HIF-1α sequence was cloned into pEX3 vector (GenePharma). pEX3-HIF-1α and its empty vector were transfected into MKN45 and SGC7901 cells. Renilla promoters were cotransfected as an internal control.
By screening the promoter region of PFKP, a binding motif (CACGC) of HIF-1α was discovered. To determine the responsive HIF-1α-binding sites in the human PFKP promoter, promoter activities were measured using Dual-Luciferase Reporter Assay System. Human full-length HIF-1α sequence was cloned into pEX3 vector (GenePharma). pEX3-HIF-1α and its empty vector were transfected into MKN45 and SGC7901 cells. Renilla promoters were cotransfected as an internal control.
7
Dual-Luciferase Reporter Assay Protocol
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Cells were transfected with pGL4.18 fused with the indicated promoters together with Renilla luciferase reporters expressing the renilla luciferase. After 48 h, renilla and firefly luciferase activities were measured by a Dual-Luciferase Reporter Assay System using a multifunctional microplate reader (Molecular Device). Renilla luciferase was used as an internal control.
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