Dual luciferase reporter plasmids containing the indicated target sites were cloned into the psiCHECK2 Vector (Promega). HeLa cells were seeded in white 96-well plates and RNAs were transfected after 8 h. There were no observable differences in cell viability between cells transfected with syn-pre-miRNAs and commercial mimics, Con or siRen. All transfections were performed in triplicates. After 24 h, plasmid DNA (20 ng/well) was transfected using jetPEI (Polyplus) according to the manufacturer's protocol. After 48 h, supernatants were removed and firefly substrate (15 µL including Lysis Buffer; Dual-Glo Luciferase Assay System, Promega) diluted with 15 µL H2O was added. Luminescence was measured on a microtiter plate reader (Mithras LB940, Berthold Technologies). After 30 min, 15 µL of Renilla substrate (including firefly Quencher Solution; Dual-Glo Luciferase Assay System, Promega) per well was added and the measurement was repeated. Values were normalized against firefly luciferase and the corresponding oligofectamine mock control, respectively.
Dual glo luciferase assay system
Manufactured by Promega
Sourced in United States, Germany, China, Switzerland, United Kingdom, Japan, Italy, Singapore
The Dual-Glo Luciferase Assay System is a reagent-based detection kit designed to quantify firefly and Renilla luciferase reporter gene activities in a single sample. The system provides a simple, sensitive, and reliable method for cell-based reporter gene analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (387 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (162 mentions)
Prl tk, by Promega (78 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (67 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (66 mentions)
Pmirglo vector, by Promega (60 mentions)
Psicheck 2 vector, by Promega (59 mentions)
Passive lysis buffer (plb), by Promega (41 mentions)
Opti mem, by Thermo Fisher Scientific (39 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (37 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (36 mentions)
Pgl3 basic vector, by Promega (34 mentions)
Glomax 20 20 luminometer, by Promega (33 mentions)
Glomax 96 microplate luminometer, by Promega (32 mentions)
Fugene hd, by Promega (32 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (31 mentions)
Glomax multi detection system, by Promega (28 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (25 mentions)
Prl sv40, by Promega (24 mentions)
Psicheck 2, by Promega (24 mentions)
2 275 protocols using dual glo luciferase assay system
1
Dual Luciferase Reporter Assay for miRNA Targeting
2
Investigating miR-221-3p Regulation of ATF3
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To investigate the relationship between miR-221–3p and ATF3, wild-type or mutant type ATF3 3′UTR (WT/MUT) including the binding sites of miR-221–3p, originated from Beyotime, then were cloned into the psiCHECK2 (Promega, Madison, WI, USA). The ATF3 3′UTR (WT/MUT) psiCHECK2 were added into 293T cells with miR-221–3p/NC mimics by using Lipofectamine 3000. Cells were harvested after 48 h post transfection and detected by the Dual-Glo Luciferase Assay System (Promega).
To verify the transcriptional regulation of GPX4 and HRD1 by ATF3, GPX4 or HRD1 promoter were cloned into the psiCHECK2. The 293T cells were co-transfected with GPX4 or HRD1 promoter vectors and oe-NC or oe-ATF3 by Lipofectamine 3000. The relative luciferase activities were tested by the Dual-Glo Luciferase Assay System (Promega).
To verify the transcriptional regulation of GPX4 and HRD1 by ATF3, GPX4 or HRD1 promoter were cloned into the psiCHECK2. The 293T cells were co-transfected with GPX4 or HRD1 promoter vectors and oe-NC or oe-ATF3 by Lipofectamine 3000. The relative luciferase activities were tested by the Dual-Glo Luciferase Assay System (Promega).
3
Investigating LINC01234 and USP4 Regulation by miR-513a-5p
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The partial sequences of LINC01234 and 3′-UTR of USP4 containing the putative binding sites of miR-513a-5p were synthetized and obtained from Sangon Biotech (Shanghai, China), then were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vectors (Promega, Madison, WI, United States) to construct wild-type reporter vectors LINC01234 (WT/MT) and USP4 (WT/MT), respectively. The LINC01234 (WT/MT) or USP4 (WT/MT) were transfected into 293T cells together with control, vector-control (NC) or miR-513a-5p agomir using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega). The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega).
4
Transcriptional Regulation of Chondrosarcoma
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SW1353 human chondrosarcoma cells were transfected with DNA using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions. After transfection of empty vector (mock), luciferase reporter, Wwp2, Wwp2-CA and Runx2 plasmids, the cells were incubated for 24 h. The cells were lysed and measured using Dual-Glo Luciferase Assay System (Promega) in a 96-well plate. For the NF-κB reporter plasmid, recombinant IL-1β (10 ng/mL) was added for 6 h before measurement. Cells were lysed and measured using Dual-Glo Luciferase Assay System (Promega) in a 96-well plate.
For knockdown experiments, siRNAs [siWwp2 (SI00223615, SI02743797), siRunx2 (SI00187915, SI02689526), or negative control (siControl) (1022076)] (QIAGEN, Valencia, CA, USA) were transfected into ATDC5 cells using Lipofectamine RNAiMAX (Life Technologies). Four hours after transfection, the cells were transfected with pNL1.1-6OSE2 or -Adamts5p using Fugene HD (Promega) according to the manufacturer’s instructions, and were incubated for 36 h. Cells were lysed and measured using Nano-Glo Luciferase Assay System (Promega) in 96-well plates.
For knockdown experiments, siRNAs [siWwp2 (SI00223615, SI02743797), siRunx2 (SI00187915, SI02689526), or negative control (siControl) (1022076)] (QIAGEN, Valencia, CA, USA) were transfected into ATDC5 cells using Lipofectamine RNAiMAX (Life Technologies). Four hours after transfection, the cells were transfected with pNL1.1-6OSE2 or -Adamts5p using Fugene HD (Promega) according to the manufacturer’s instructions, and were incubated for 36 h. Cells were lysed and measured using Nano-Glo Luciferase Assay System (Promega) in 96-well plates.
5
STAT3 Luciferase Reporter Assay
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Cells were seeded in 96-well plates at a density of 1×104 cells/well and transiently co-transfected with a mixture of Renilla plasmid and the STAT3 luciferase reporter construct using Lipofectamine 2000 reagent (Invitrogen) on the next day. The cells were lysed in passive lysis buffer provided by Dual-Glo Luciferase Assay System (Promega) 48h after transfection. The Renilla and firefly luciferase luminescence were measured using the Dual-Glo Luciferase Assay System (Promega).
6
DENV Modulation of NF-κB Signaling
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HEK 293T cells were plated in 96 well plates and were co-transfected with a NF-κB luciferase reporter, a Renilla luciferase reporter, and a plasmid encoding Flag-tagged RIPK1 using GeneJuice® Transfection Reagent (EMD Millipore), according to the manufacturer’s protocol. Co-transfected cells were infected with DENV2 (NGC, MOI=3) and were cultured for 72hrs or transfected with plasmids expressing DENV NS3, NS2B3, NS2B3-S135A, or NS1 and cultured for 48hrs. Cells were then stimulated with hTNFα (50ng/ml, Peprotech) for the final 6hrs. Luciferase activity was quantified using the dual Glo luciferase assay system (Promega). HEK 293T cells stably expressing TLR3 were co-transfected and infected with DENV2 or overexpressed DENV NS3, NS2B3, NS2B3-S135A, or NS1 as described above and were stimulated with poly I:C (20µg/ml) for the final 24hrs. At the end of the treatment, luciferase activity was measured using the dual Glo luciferase assay system (Promega). The luciferase signal for each well was normalized by the Renilla luciferase signal.
7
Investigating 3'-UTR and 5'-UTR Effects on Gene Expression
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To evaluate the effect of 3′-UTR on HRAS expression, the wild type or mutant of 3′-UTR of HRAS was inserted behind the CDS region of the firefly luciferase (F-luc). Both vectors were transfected into wild type or METTL3-Mut/- PCa cells for 24 h, the firefly luciferase (F-luc) and Renilla luciferase (R-luc) were assayed by Dual-Glo Luciferase Assay system (Promega). Similarly, to evaluate the potential roles of 5′-UTR in MEK2 expression, the wild type or mutant 5′UTR ligated with promoter of MEK2 was inserted in the front of the F-luccoding region of the plasmid to generate MEK2-5′UTR, MEK2-5′UTR-Mut, respectively. Both plasmids were transfected into wild-type or METTL3-Mut/- PCa cells for 24 h. The Firefly luciferase (F-luc) and Renilla luciferase (R-luc) were assayed by Dual-Glo Luciferase Assay system (Promega). Specific steps of Dual-luciferase assay according to described protocol. Renilla luciferase (R-luc) was used to normalize Firefly luciferase (F-luc) activity. Experiments were carried out for three times to minimize the experimental bias.
8
Luciferase Assay for miRNA Targets
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HEK293T cells in collagen-coated 24-well plates (Iwaki) were co-transfected with 5 ng of pGL3-hCMV/CAP1 3′UTR (either Wild-type, Single mut., or Triple mut.), 5 ng of pRL-TK (Promega), and 790 ng of either empty plasmid (pUC18 or pcDNA3.1[+]) or each miRNA expression plasmid, as indicated in Fig. 4 B, in triplicate using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection and subjected to the luciferase assay with the Dual-Glo Luciferase Assay System (Promega) using Lumat LB9507 (Berthold). The significance of differences was assessed using the Student’s t-test.
Uninfected TE671 and TE671/SFVmfu(PI) cells in collagen-coated 24-well plates (Iwaki) were co-transfected with 80 ng of pGL3-hCMV/CAP1 3′UTR (either Wild-type, Single mut., or Triple mut.), or pGL3-hCMV, 80 ng of pRL-TK, and 640 ng of the empty plasmid (pcDNA3.1[+]) in triplicate using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection and assayed for luciferase activity with the Dual-Glo Luciferase Assay System (Promega) using Lumat LB9507 (Berthold). The significance of differences was assessed using the Student’s t-test.
Uninfected TE671 and TE671/SFVmfu(PI) cells in collagen-coated 24-well plates (Iwaki) were co-transfected with 80 ng of pGL3-hCMV/CAP1 3′UTR (either Wild-type, Single mut., or Triple mut.), or pGL3-hCMV, 80 ng of pRL-TK, and 640 ng of the empty plasmid (pcDNA3.1[+]) in triplicate using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection and assayed for luciferase activity with the Dual-Glo Luciferase Assay System (Promega) using Lumat LB9507 (Berthold). The significance of differences was assessed using the Student’s t-test.
9
Investigating miR-30b and MRPL23-AS1 Regulation of Wnt/β-catenin Signaling
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OS cells (1 × 105) were seeded into 6-well plates, grown overnight, and then co-transfected with miR-30b mimics and MRPL23-AS1 or MYH9 3`-UTR pmiR-GLO luciferase construct using Lipofectamine™ 2000 for 24 h. Furthermore, to assess the MRPL23-AS1-mediated CTNNB1 transcription in activation of the Wnt/β-catenin signaling pathway, the TOP/FOP-flash assay was used by transfecting OS cells with TOP-flash (the TCF reporter plasmid; Merck Millipore, Schwalbach, Germany) or FOP-flash (plasmid carrying the mutant TCF binding sites; Merck-Millipore) using Lipofectamine™ 2000 for 24 h, while the renilla luciferase reporter plasmid pR-TK (Promega, Madison, WI, USA) was used as the control. The luciferase activity was measured by using the Promega Dual-Glo® luciferase assay system (Promega) as per the supplier’s protocols.
10
Assessing CMV Enhancer Activity in Zebrafish
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To assess the activity of the CMV enhancer in zebrafish cell culture, 4000ng of firefly luciferase expressing plasmids either with or without the CMV enhancer were transfected into 2x10 6 PAC2 cells via electroporation (Fig. 1A). 100ng of the renilla luciferase expressing plasmid (pRL-SV40 Promega, E2231) was included as a transfection control. Firefly/renilla luciferase signal was calculated as the mean of ratios of three technical replicates per biological replicate. Fold change was then calculated relative to the signal of the SV40 promoter-only containing plasmid. The mean of fold-changes is reported and error bars represent standard deviation.
To test the functionality of our dual module lac repressor system in zebrafish cell culture, 2000ng
repressible module and 2000ng of LacI-expressing plasmid were co-transfected into 2x10 6 PAC2 cells by electroporation. 400ng of pRL-SV40 was included as a transfection control (Fig. 2).
All transfections were completed using 2mM cuvettes (Bulldog Bio, 12358-346) and electroporated using a NEPA21 Electroporator (Nepagene). Cells were harvested from culture and resuspended in 90μL of Opti-MEM Reduced Serum Medium (ThermoFisher, 31985062) per Luciferase results were collected 48 hours post-transfection on a GloMax-Multi+ Detection System (Promega, E7081) using the Promega Dual-Glo Luciferase Assay System (Promega, E2940).
To test the functionality of our dual module lac repressor system in zebrafish cell culture, 2000ng
repressible module and 2000ng of LacI-expressing plasmid were co-transfected into 2x10 6 PAC2 cells by electroporation. 400ng of pRL-SV40 was included as a transfection control (Fig. 2).
All transfections were completed using 2mM cuvettes (Bulldog Bio, 12358-346) and electroporated using a NEPA21 Electroporator (Nepagene). Cells were harvested from culture and resuspended in 90μL of Opti-MEM Reduced Serum Medium (ThermoFisher, 31985062) per Luciferase results were collected 48 hours post-transfection on a GloMax-Multi+ Detection System (Promega, E7081) using the Promega Dual-Glo Luciferase Assay System (Promega, E2940).
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