H358, ER-HRASG12V MCF10A and KP (tetON) cells were plated in 96 well plates and the following day co-transfected with pRL-TK control and pGL3-3′UTR PD-L1 luciferase constructs using Lipofectamine 2000 (Life Technologies). 24 h after transfection, PMA (200 nM; Sigma), doxycycline (1 μg/ml; Sigma) or MEK inhibitor GSK1120212 (25 nM; Selleckchem) was added, and 6-7 h later the Dual-Luciferase Reporter Assay (Promega) was performed. For ER-HRASG12V MCF10A, 24 h after transfection cells were serum-starved overnight, and then treated with 4-OHT (100 nM) for 24 h before the Dual-Luciferase Reporter Assay (Promega) was performed.
Dual luciferase reporter assay
Manufactured by Promega
Sourced in United States, Germany, China, United Kingdom, France, Switzerland, Japan
The Dual-Luciferase Reporter Assay is a laboratory equipment product that measures the activity of two different luciferase reporter enzymes simultaneously. It provides a method for analyzing gene expression, signal transduction pathways, and other biological processes in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (266 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (86 mentions)
Passive lysis buffer (plb), by Promega (84 mentions)
Prl tk, by Promega (51 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (47 mentions)
Psicheck 2 vector, by Promega (33 mentions)
Glomax 20 20 luminometer, by Promega (25 mentions)
Pgl3 vector, by Promega (23 mentions)
Pmirglo vector, by Promega (23 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (21 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (20 mentions)
Pgl3 control vector, by Promega (20 mentions)
Pgl3 basic vector, by Promega (20 mentions)
Psicheck 2, by Promega (20 mentions)
Dual luciferase reporter assay system, by Promega (20 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (19 mentions)
Prl tk vector, by Promega (17 mentions)
Pmir report vector, by Thermo Fisher Scientific (14 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (14 mentions)
Opti mem, by Thermo Fisher Scientific (13 mentions)
1 553 protocols using dual luciferase reporter assay
1
Regulation of PD-L1 3'UTR Activity
2
Probing AR-Dependent Transcriptional Activity
3
Quantitative Dual Luciferase Reporter Assay
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Luciferase activities were measured by using the dual luciferase reporter assay (Promega) according to the manufacturer's protocol. pRL/TK-luciferase reporter plasmid was used as a second reporter. The data were obtained by analyzing triplicated samples. In general, 100 ng expression plasmid, 60 ng 8xGTIIC-luciferase (Addgene), and 3 ng pRL-TK (internal control) were co-transfected into H1299 cells plated in 24-well plates. 48 h later, cells were harvested and luciferase activities were measured by using the dual luciferase reporter assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. All reporter assays were completed at least in triplicate, and the results were shown as average values ± standard deviations (SD) from one representative experiment.
4
Probing AR-Dependent Transcriptional Activity
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To determine AR-dependent transcriptional activity, HEK293T cells were
transiently transfected in culture media containing 10% charcoal-dextran
stripped serum with ARE-firefly luciferase and CMV-Renilla luciferase (Cignal
ARE Reporter Assay Kit, Qiagen), in addition to either FLAG-AR24Q or FLAG-AR65Q
(gift from Maria Pennuto). Following 24 h of transfection, cells were washed and
treated with vehicle, bicalutamide, TA, or MEPB for 24 h. Firefly and Renilla
luciferase substrates (Dual-Luciferase Reporter Assay, Promega) were added, and
luciferase activity was measured using a microplate spectrophotometer (BioTek).
Mammalian two-hybrid assays were performed by transiently transfecting HEK293T
cells with pG5Luc firefly luciferase reporter (Checkmate Mammalian Two-Hybrid
kit, Promega), CMV-Renilla luciferase (Cignal ARE Reporter Assay Kit, Qiagen),
and GAL4 DBD-AR LBD (gift from Elizabeth Wilson), in addition to either VP16
empty vector, VP16-NCoR, or VP16-SMRT (gifts from Vivian Bardwell) for 24 h.
Cells were then washed and treated with vehicle, TA, or MEPB for 24 h. Renilla
and firefly luciferase were quantified using Dual-Luciferase Reporter Assay
(Promega) and a microplate spectrophotometer (BioTek).
transiently transfected in culture media containing 10% charcoal-dextran
stripped serum with ARE-firefly luciferase and CMV-Renilla luciferase (Cignal
ARE Reporter Assay Kit, Qiagen), in addition to either FLAG-AR24Q or FLAG-AR65Q
(gift from Maria Pennuto). Following 24 h of transfection, cells were washed and
treated with vehicle, bicalutamide, TA, or MEPB for 24 h. Firefly and Renilla
luciferase substrates (Dual-Luciferase Reporter Assay, Promega) were added, and
luciferase activity was measured using a microplate spectrophotometer (BioTek).
Mammalian two-hybrid assays were performed by transiently transfecting HEK293T
cells with pG5Luc firefly luciferase reporter (Checkmate Mammalian Two-Hybrid
kit, Promega), CMV-Renilla luciferase (Cignal ARE Reporter Assay Kit, Qiagen),
and GAL4 DBD-AR LBD (gift from Elizabeth Wilson), in addition to either VP16
empty vector, VP16-NCoR, or VP16-SMRT (gifts from Vivian Bardwell) for 24 h.
Cells were then washed and treated with vehicle, TA, or MEPB for 24 h. Renilla
and firefly luciferase were quantified using Dual-Luciferase Reporter Assay
(Promega) and a microplate spectrophotometer (BioTek).
5
Dual-Luciferase Assay for miR-485-5p Targets
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miR-NC or miR-485-5p mimics was co-transfected with SNHG7 mut or SNHG7 wt into HEK-293T cells for 48 h. Dual-luciferase reporter assay (Promega) was carried out according to the protocol. Similarly, miR-NC or miR-485-5p mimics was co-transfected with JUND mut or JUND wt, and Dual-luciferase reporter assay (Promega, USA) was conducted subsequently.
6
miR-503 Regulation of PDCD4 Expression
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For the 3′ UTR luciferase reporter assay, the miR-503 mimics or inhibitor and pmirGLO, pmirGLO-PDCD4 3′ UTR-wt, pmirGLO-PDCD4–3′ UTR-mut were cotransfected into HEK293T. Cell lysates were collected at 48 hours post-transfection. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega). For the luciferase promoter assay, different segments of the miR-503 promoter were produced by PCR using primers and were cloned into pGL3 vectors. The potential binding sites for TGF-b were mutanted for site-specific mutagenesis. U251 cells were transfected with pGL-miR-503-wt-set4, pGL-miR-503-mut-SBE1, pGL-miR-503-mut-SBE2, pGL-miR-503-mut-SBE3, or pGL-miR-503-mut-SBE4 and Renilla. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega).
7
Detecting miR-195 and SIRT1 Interaction
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A dual-luciferase reporter gene was used to detect the targeted relationship between miR-195 and SIRT1. HRECs and HMECs were seeded in a 96-well plate and cells in the miR-195 inhibitor group, SIRT1-Mut and SIRT1-Wt were transfected with Lipofectamine TM3000. All constructs were verified by sequencing. The underlined nucleotides (MI0000489) indicated the bases where mutations were made. According to the manufacturer's instructions, the luciferase activities of fireflies and sea kidneys were analyzed using the Dual-Luciferase Reporter Assay (Promega). For luciferase assay, 293A cells were cultured in 24-well plates and transfected with 100 ng luciferase reporter plasmid, 5 ng pRL-TK vector expressing the Renilla luciferase (Promega), and 50 pmol/L of miR-195 inhibitor group, SIRT1-Mut, SIRT1-Wt, or negative control precursor. After transfection for 36 h, firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay (Promega). Each transfection was repeated twice in triplicate.
8
Overexpression and Luciferase Assay
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The overexpression and luciferase reporter plasmids were constructed by GeneChem Co., Ltd (Shanghai, China). These plasmids were transfected into HEK-293T cells by Effectene Transfection Reagent (Qiagen, Hilden, Germany) as described in the Supplementary Information . The relative luciferase activity was analyzed by the Dual Luciferase Reporter (DLR) Assay (Promega, Madison, WI, USA).
9
Construction and Luciferase Assay of Vang Sensor
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To construct the wild-type Vang sensor, we amplified the 3’UTR of Vang using the PCR primers (NotI_Vang_2128 and XhoI_Vang_3492, Supplementary file 4 ) and genomic DNA as a template, and the fragment was inserted to the Not I and Xho I sites of the modified psiCHECK2 plasmid (Okamura et al., 2007 (link)). The mutant sensor was constructed by overlapping PCR using the mutagenesis primers shown in Supplementary file 4 . Luciferase assays were performed using S2-R+ cells according to the previously published protocol (Okamura et al., 2007 (link)). Briefly, S2-R+ cells were seeded at 1 × 10^6 cells/ml in a 96-well plate, and cells were transfected using Effectene (Qiagen). 25 ng of the sensor plasmid, 25 ng of the miRNA expression plasmid and 12.5 ng of the Ub-Gal4 plasimid was used for each well. Dual-Luciferase Reporter (DLR) assay (Promega, USA) was carried out to quantitate the effects of miR-3 overexpression on the respective Vang sensors. The assay was performed according to the manufacturer's protocol. Assays were performed in quadruplicate at each time, and repeated twice on different days. The two sets of data were combined to draw the charts.
10
Dual-Luciferase Reporter Assay for Gene Expression
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The dual-Luciferase reporter (DLR) assay by Promega was used for quantification of FLuc and RLuc activities. The cells were collected from a plate well into an Eppendorf tube 30 h post-transfection, pelleted at 830 rcf for 1 min, washed with PBS buffer, and pelleted again. The pelleted cells were resuspended in 115 μl of a freshly made passive lysis buffer (DLR, Promega, Madison, WI, USA), lysed for 20 mins while shaking, and frozen at −80°C. Standard protocols were then used to determine activities of both luciferases using the GloMax 20/20 luminometer (Promega, Madison, WI, USA). FLuc/RLuc relative activities were normalized to that of a control and averaged for two technical replicates. An average ± one-standard deviation was calculated for normalized fold differences from at least four separate transfections.
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