The wild-type or mutant PCGEM1 was cloned into pmirGLO plasmid (named pmirGLO-PCGEM1 and pmirGLO-PCGEM1-mut). Cells were co-transfected with pmirGLO-PCGEM1 or pmirGLO-PCGEM1-mut and miR-NC or miR-182. After 48 h, Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). To detect the effect of PCGEM1 on FBXW11 3′UTR, the FBXW11 3′UTR was cloned into pmirGLO plasmid (named pmirGLO-FBXW11). Cells were co-transfected with pmirGLO-FBXW11 and miR-NC or miR-182. After 48 h, Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). NF-κB and β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter plasmid encoding Renilla luciferase was purchased from Promega. Cells were plated in a 24-well plate and cotransfected with PCGEM1, NF-κB or β-catenin/TCF firefly luciferase reporter, and pRL-TK (Promega) by using Lipofectamine 2000. The pRL-TK vector was used as an internal control. 48 h later, cells were collected and analyzed using the Dual-Luciferase Reporter Assay System (Promega).
Dual luciferase reporter assay system
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The Dual-Luciferase Reporter Assay System is a laboratory tool designed to measure and compare the activity of two different luciferase reporter genes simultaneously. The system provides a quantitative method for analyzing gene expression and regulation in transfected or transduced cells.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2902 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (992 mentions)
Passive lysis buffer (plb), by Promega (531 mentions)
Prl tk, by Promega (470 mentions)
Psicheck 2 vector, by Promega (426 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (390 mentions)
Pmirglo vector, by Promega (368 mentions)
Pgl3 basic vector, by Promega (336 mentions)
Glomax 20 20 luminometer, by Promega (261 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (247 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (245 mentions)
Pgl3 vector, by Promega (226 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (203 mentions)
Psicheck 2, by Promega (144 mentions)
Prl sv40, by Promega (143 mentions)
Pgl3 basic, by Promega (131 mentions)
Prl tk vector, by Promega (126 mentions)
Pgl3 control vector, by Promega (126 mentions)
Pmirglo, by Promega (124 mentions)
Prl tk plasmid, by Promega (121 mentions)
15 923 protocols using dual luciferase reporter assay system
1
PCGEM1 Regulation of FBXW11 and Transcriptional Pathways
2
Dual Luciferase Assay for Transfected Protoplasts
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Protoplasts that had been transfected with 10 μg of each individual construct or equivalent volume of water for the negative controls were used for the dual luciferase assay. 16–24 hours after transfection, protoplasts were pelleted by centrifugation (14,000 x g for 30 seconds). Protoplasts were lysed in 1x passive lysis buffer (the Dual-Luciferase® Reporter Assay System, Promega) and incubated at room temperature for 15 minutes. Following the incubation, 20μL (approximately 6.6x 104 cells) of lysed protoplasts were added to 100 μL LARII (the Dual-Luciferase® Reporter Assay System, Promega) vortexed briefly and measured immediately in a luminometer (Sirius Luminometer, Berthold Detection Systems). The luminescence of Luciferase was quenched and Renilla luminescence measured by the addition of 100 μL of Stop & Glo® Buffer (the Dual-Luciferase® Reporter Assay System, Promega). Luminescence was measured in technical triplicates for all combinations of transfected plasmids and each transfection was repeated thrice, statistical analysis performed using GraphPad Prism.
3
Luciferase Assay for miR-181b and SP1 Binding
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A luciferase reporter assay was used to detect the binding of miR-181b to SP1. Briefly, the SP1 3′-UTR are cloned into the SacI and HindIII sites of the pmiRNA-Report vector (Genechem, Shanghai, China) and confirmed by a sequencing to form the wild-type (WT) and mutated (Mut). Cells, which co-transfected with the WT or Mut reporter plasmid, pRLTK plasmid, and the miR-181b mimic or miR-NC were seeded in the 24-well plate. After transfection 24 h, promega Dual Luciferase Reporter Assay System (Promega) was performed to measure the luciferase activity.
To identify the regulation of SP1 on PKM2 and Glut1 promoter region, U87 and U251 cells were seeded in 24-well plate at a density of 6 × 104 cells per well for 24 h before transfection. The cells were co-transfected with a mixture of luciferase reporter vectors (pGL3-basic) containing wild type sequence or mutant sequence of specific PKM2 and Glut1 promoter fragment. After 48 h, the luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
To identify the regulation of SP1 on PKM2 and Glut1 promoter region, U87 and U251 cells were seeded in 24-well plate at a density of 6 × 104 cells per well for 24 h before transfection. The cells were co-transfected with a mixture of luciferase reporter vectors (pGL3-basic) containing wild type sequence or mutant sequence of specific PKM2 and Glut1 promoter fragment. After 48 h, the luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
4
Luciferase Assay for Transcriptional Activity
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MEFs were co-transfected with a tk-Renilla plasmid that allows the normalization of transfection efficiency. MEFs were lysed in Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, Promega) and shaken for 15 min. Lysates were used for measurement of Luciferase and Renilla activities (Dual-Luciferase Reporter Assay System, Promega). The ratio between Luciferase and Renilla activities was further used to compare the induction of Luciferase activity between samples. Triplicates (n = 3) were obtained for the experiments presented in Figure 4 , Supplementary Figures S5 and S6. Two to four experiments (N = 2–4) were performed in triplicate for the experiments presented in Figure 6 and Supplementary Figure S7.
5
Quantifying NF-κB Activation via Luciferase Assay
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A dual luciferase reporter assay was performed as described previously (Li et al., 2007 (link)). The Dual-Luciferase® Reporter Assay System (Promega, Madison, United States) was used to detect NF-κB activation using a GloMax® 20/20 tube luminometer (Promega, United States). Briefly, RAW264.7 cells were co-transfected with pGL3-NF-κB-luc, Renilla, pcDNA3.1-Rv1768, or pcDNA3.1. Then, 24 h post-transfection, cells were treated with LPS (1000 ng/mL, Sigma-Aldrich) to stimulate NF-κB activation. After 1 h, the cells were collected. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). Data were normalized for transfection efficiency by dividing firefly luciferase activity with that of Renilla luciferase.
6
Dual Luciferase Promoter Assay
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Cells were grown in 24-well plates for 24 h to 70–90% confluence. Next, 0.5 μg of the pGL3-basic plasmid containing the promoter and 2.5 ng of the pRL-CMV plasmid were transfected into cells with 1.5 μL of Lipofectamine 3000 (Invitrogen). The transfection reagent was removed, and then 500 μL of medium with 10% FBS was added to each well after 5 h. After 24 h, the cells were lysed, and the luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) on a Turner Designs TD-20/20n luminometer (Promega). The Dual Luciferase Reporter Assay System was purchased from Promega (Madison, WI, United States). The ratio of the firefly luciferase activity to the Renilla luciferase activity represents the strength of the promoter.
7
Regulation of MAVS and MARL by miR-122
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The MARL wild type and the mutant devoid of miR-122 binding site was contransfected with miR-122 mimics into EPC cells. At 48 h post-transfection, reporter luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega). To determine the functional regulation of MAVS or MARL, MIC cells were cotransfected MAVS expression plasmid or MARL expression plasmid, together with NF-κB, IRF3, IFN-1, and IFN-2 luciferase reporter gene plasmids, pRL-TK Renilla luciferase plasmid, either miR-122 mimics or negative controls. At 48 h post-transfection, the cells were lysed for reporter activity using the Dual-Luciferase reporter assay system (Promega). miR-122 sensor was cotransfected with miR-122 mimics or MARL expression plasmid into MIC cells. At 48 h post-transfection, the cells were lysed for reporter activity. All the luciferase activity values were achieved against the renilla luciferase control. Transfection of each construct was performed in triplicate in each assay. Ratios of renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged.
8
Deciphering HOTAIR-miR-106a-5p and miR-106a-5p-STAT3 Interactions
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StarBase v2.0 (http://starbase.sysu.edu.cn ) online database was used to predict
the interactions between HOTAIR and miR-106a-5p. The sequence of HOTAIR
containing a miR-106a-5p binding site was amplified and cloned to psiCHECK-2
vector (Promega, Madison, WI, USA) to generate HOTAIR-WT (wild-type). The
binding site of HOTAIR was mutated to obtain the HOTAIR-MUT (mutant type) using
a Site-Directed Mutagenesis Kit (Stratagene, South Carolina, USA). HOTAIR-WT (or
HOTAIR-MUT) and miR-106a-5p mimic were cotransfected into HEK293A cells, and
relative luciferase activities were measured using the Dual-Luciferase Reporter
Assay System (Promega).
TargetScan Human 7.2 (http://www.targetscan.org/vert_72/ ) online database was used to
predict the interactions between miR-106a-5p and STAT3. Wild-type and three
mutant-type STAT3 reporters containing miR-106a-5p binding sites were amplified
and cloned into psiCHECK-2 vector to obtain STAT3-WT, STAT3-MUT1, STAT3-MUT2,
and STAT3-MUT Both. STAT3-WT, STAT3-MUT1, STAT3-MUT2, or STAT3-MUT both were
transfected into HEK293A cells together with miR-106a-5p mimic, and relative
luciferase activities were measured using the Dual-Luciferase Reporter Assay
System (Promega).
the interactions between HOTAIR and miR-106a-5p. The sequence of HOTAIR
containing a miR-106a-5p binding site was amplified and cloned to psiCHECK-2
vector (Promega, Madison, WI, USA) to generate HOTAIR-WT (wild-type). The
binding site of HOTAIR was mutated to obtain the HOTAIR-MUT (mutant type) using
a Site-Directed Mutagenesis Kit (Stratagene, South Carolina, USA). HOTAIR-WT (or
HOTAIR-MUT) and miR-106a-5p mimic were cotransfected into HEK293A cells, and
relative luciferase activities were measured using the Dual-Luciferase Reporter
Assay System (Promega).
TargetScan Human 7.2 (
predict the interactions between miR-106a-5p and STAT3. Wild-type and three
mutant-type STAT3 reporters containing miR-106a-5p binding sites were amplified
and cloned into psiCHECK-2 vector to obtain STAT3-WT, STAT3-MUT1, STAT3-MUT2,
and STAT3-MUT Both. STAT3-WT, STAT3-MUT1, STAT3-MUT2, or STAT3-MUT both were
transfected into HEK293A cells together with miR-106a-5p mimic, and relative
luciferase activities were measured using the Dual-Luciferase Reporter Assay
System (Promega).
9
Elucidating SNHG22-miR-200c-3p Interaction
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To confirm that SNHG22 could sponge miR-200C-3p, HEK-293T cells were co-transfected with the mixture of luciferase reporter vectors (pmirGLO) containing SNHG22-miR-200c-3p binding sequences or mutant sequences and miRNA mimics (20 nM) to examine the interaction between SNHG22 and miR-200c-3p. A dual luciferase reporter assay system (Promega, Madison, WI, USA) was employed to measure the luciferase activity according to the manufacturer’s protocol.
To explore the transcriptional regulation of ELK4 on SNHG22, HEK-293T cells were co-transfected with luciferase reporter comprising wild type or mutant SNHG22 promoter region and empty vector or ELK4 plasmid (Genechem). A dual luciferase reporter assay system (Promega, Madison, WI, USA) was employed to measure the luciferase activity according to the manufacturer’s protocol.
To explore the transcriptional regulation of ELK4 on SNHG22, HEK-293T cells were co-transfected with luciferase reporter comprising wild type or mutant SNHG22 promoter region and empty vector or ELK4 plasmid (Genechem). A dual luciferase reporter assay system (Promega, Madison, WI, USA) was employed to measure the luciferase activity according to the manufacturer’s protocol.
10
Assessing LINC01134 Regulation of AKT1S1 and NF-κB
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To assess the effects of LINC01134 on AKT1S1 promoter activity, the AKT1S1 promoter reporter pGL3-AKT1S1 was co-transfected with pRL-TK and LINC01134 overexpression or silencing vectors into HCCLM3 cells. pRL-TK encodes renilla luciferase and was used as an endogenous reference. Forty-eight hours after transfection, the firefly luciferase and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). To assess the effects of LINC01134 on NF-κB transcriptional activity, firefly luciferase reporter containing NF-κB binding sites (pNFκB-luc) (Beyotime) was co-transfected with pRL-TK and LINC01134 overexpression or silencing vectors into HCCLM3 cells. Forty-eight hours after transfection, the firefly luciferase and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).
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