Oligonucleotides containing the regulatory elements overlapping candidate CAD loci (156 nt for 1 × construct or 468 nt for 3 × construct) were synthesized for each allele and cloned into a shuttle vector pUC57 (GeneWiz). These fragments were then isolated by double restriction digest and subcloned into the multiple cloning site of the minimal promoter containing pLuc-MCS luciferase reporter vector (Agilent). All constructs were validated by Sanger sequencing. Empty vector (pLuc-MCS), 1 × and 3 × CAD loci and Renilla luciferase constructs were co-transfected, with or without pcDNA3-empty, pcDNA3-JUN, JUNB or JUND expression constructs into A7r5 or HCASMCs using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. For siRNA knockdown studies, 20 nM Silencer Select siRNA negative control or siRNA against JUN, JUNB or JUND (Life Technologies) was transfected in HCASMCs 12 h before reporter transfections using RNAiMAX (Life Technologies). Media was changed after 6 h, and dual-luciferase activity (Promega) was recorded after 24 h using a SpectraMax L luminometer (Molecular Devices). Relative luciferase activity (firefly/Renilla luciferase ratio) is expressed as the fold change of the empty vector control (pLuc-MCS).
Dual luciferase activity
Manufactured by Promega
Sourced in United States
The Dual-luciferase activity is a laboratory instrument used to measure the activity of two different luciferase reporter genes simultaneously. It provides a quantitative analysis of gene expression or regulatory pathways.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax l luminometer, by Molecular Devices (3 mentions)
Wallac victor v 1420 multilabel counter, by PerkinElmer (2 mentions)
Spectramax l, by Molecular Devices (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Puc57, by Genewiz (1 mentions)
Psicheck2 dual luciferase vector, by Promega (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Living image software 4, by PerkinElmer (1 mentions)
Glomax, by Promega (1 mentions)
Ivis lumina xrms imaging system, by PerkinElmer (1 mentions)
Il 1β, by Promega (1 mentions)
18 protocols using dual luciferase activity
1
Regulatory Loci Modulation of CAD Expression
2
Dual-luciferase Assay for CD24 Promoter Activity
3
Luciferase Assay for SLC1A2 3' UTR Regulation
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The SLC1A2-3’ UTR WT was PCR-amplified directly from mouse cDNA and then cloned into the 3’ end of the Renilla luciferase gene of the psiCHECK-2 dual-luciferase vector containing firefly luciferase driven by a different promoter as an internal control (Promega, Madison, WI, USA) via the Xho I and Not I sites. Mutant reporter constructs (SLC1A2-3’ UTR-A, SLC1A2-3’ UTR-C) were obtained from the SLC1A2-3’ UTR WT reporter using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primary astrocytes were cultured, and transfection using Lipofectamine 3000 reagents (Life Technologies), and dual-luciferase activity (Promega) was measured with the Wallac Victor V 1420 Multilabel Counter (PerkinElmer, San Jose, CA, USA) to yield the ratio of Renilla luciferase activity to firefly luciferase activity. All luciferase readings were taken from more than three individual biological repeats.
4
Luciferase reporter assay for NF-κB activity
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Wild-type or N4BP1−/− HEK293T cells seeded on 24-well plates were transiently transfected with 100 ng of the luciferase reporter plasmid together with a total of 800 ng of various expression plasmids or empty control plasmids. As an internal control, 10 ng of pRL-SV40 was transfected simultaneously. 24 h after transfection, the cells were stimulated with IL-1β (50 ng/ml) for 8 h. Dual luciferase activity in the total cell lysates was quantified (Promega).
5
Measuring Neuronal Transcriptional Activity
6
Luciferase Assay for MFAP5 Regulation
7
Transient Protoplast Transformation and Dual-Luciferase Assay
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PEG-Ga solution (40% PEG 4000, 0.2 M Mannitol, 0.1 M GaCl2) was used by protoplasts transformation. The single sample needed 110 μL PEG-Ga solution, and this transformation reaction lasted six minutes before dilution with 440 μL MMG (15 mM MgCl2, 0.4 M Mannitol, 4 mM MES). Protoplasts after transformation reaction were washed by WI (0.5 M Mannitol, 4 mM MES, 20 mM KCl) for dual-Luciferase assay. The C4-NADP-ME (Zm00001eb121470) promoter sequence was recombined with the PBI221 vector, in which the GUS (β-glucuronidase) reporter gene was replaced with the LUC (fireflyluciferase) reporter gene [43 (link)]. The coding sequences of bHLH157 and NF-YC2 were cloned into the PBI221 vector, and the two recombinant vectors were co-transformed into maize leaf protoplasts to evaluate the relative activity of LUC. Dual-luciferase activity (Promega) was measured to verify the interactions between bHLH157 and/or NF-YC2 and C4 NADP-ME. GUS activity was used as an internal reference to normalize the transformation efficiency of protoplasts, and the relative ratio of LUC/GUS (4 h–0 h) between the experimental group and control was used to represent the relative activity of the promoter [44 (link)].
8
Transcriptional activity of SAMD12-AS1
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HepG2 cells were plated in 24-well plates and cotransfected with the pGL2-SAMD12-AS1-promoter and pRL-TK as an internal control and pCMV FLAG-HBx or pCMV FLAG-HBc. The cell lysates were harvested, and dual-luciferase activity was assessed according to the manufacturer’s instructions (Promega, USA). Relative luciferase activity was calculated (Firefly luciferase/Renilla luciferase).
9
Dual-luciferase Assay of miR-346
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SMMC-7721 cells (4 × 105) were seeded in six-well plates and cotransfected with the reporter constructs together with the miR-346 mimic or NC using Lipofectamine 2000 according to the manufacturer’s recommendation (Invitrogen). Renilla luciferase was used for endogenous normalization. Dual-luciferase activity was determined after 48 h according to the manufacturer’s instructions (Promega, Beijing, P.R. China).
10
Dual-Luciferase Assay for NF-κB Pathway
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HEK-293FT cells were seeded in 12-well format and both pdCas9-Tet1-CD and pcDNA3.1-MS2-Tet1-CD plasmids were transfected using polyethylenimine reagents. Two days later, cells were seeded into 12-well plates. At day 3, pHAGE-RANK-EGFP, pGL3-NF-κB and pRL-TK plasmids were co-transfected using polyethylenimine reagents. Twenty-four hours later, dual-luciferase activity was measured following the manufacturer’s instruction (Promega, Madison, WI, USA).
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