The WT and MT KDM4A 3′-UTR were cloned into pMIR as the 3′-UTR to luciferase. The pMIR-3′-UTR constructs and a β-galactosidase construct for normalization were co-transfected with the indicated miRNA mimics for 48 h using X-tremeGENE siRNA transfection reagent (Roche Applied Science) in Opti-MEM I media (Life Technologies, Inc.). Cells were collected by scraping, and lysates were prepared following the Dual-Light system instructions (Life Technologies, Inc.). The Dual-Luciferase and β-galactosidase assays were performed using the Dual-Light system following the manufacturer's instructions (Life Technologies, Inc.). Measurements for two biological replicates were taken in triplicate and averaged.
Dual light system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dual-Light System is a laboratory equipment designed to perform luminescence-based assays. It can be used to detect and measure both bioluminescence and chemiluminescence in biological samples. The system provides a reliable and sensitive platform for various applications, such as cell-based assays, reporter gene studies, and protein-protein interaction analyses.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Pmir report beta gal vector, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pmir report luciferase plasmid, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pmir report vector, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Lysis solution, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Opti mem 1 media, by Thermo Fisher Scientific (1 mentions)
X tremegene sirna transfection reagent, by Roche (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Six well cell culture plates, by BD (1 mentions)
Jetprime reagent, by Polyplus Transfection (1 mentions)
Lipofectamine 2000 system, by Thermo Fisher Scientific (1 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
36 protocols using dual light system
1
Dual-Luciferase Assay for miRNA Targets
2
Sall4 Regulation of Col1a1 and Col12a1
3
Investigating miR-219 Regulation of Sall4
4
Sall4 Regulation of Col1a1 and Col12a1
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To determine if Sall4 could functionally regulate transcription of Col1a1 or Col12a1, HEK293T cells were plated at a density of 2×104 cells per well in a 96-well plate coated with poly-D -lysine and allowed to adhere overnight. HEK293T cells were then co-transfected with either 112.5 ng per well of pGL3 Enhancer Col1a1 Intron 1, 37.5 ng per well pMIR-Report beta gal vector (Life Technologies) and 50 ng per well pcDNA3.1 MCS-BirA(R118G)-HA (with or without axolotl Sall4 insert) or 125 ng per well of pGL3 Enhancer Col12a1 Intron 1, 25 ng per well pMIR-Report beta gal vector (Life Technologies) and 50 ng per well pcDNA3.1 MCS-BirA(R118G)-HA (with or without axolotl Sall4 insert) using Lipofectamine 3000 (Invitrogen). Cells were incubated for 48 h and luminescence was detected using the Dual Light System (Ambion) following the manufacturer’s protocol.
5
Validating Sall4 as miR-219 Target
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To determine if Sall4 is a target of miR-219, HEK 293 cells were plated in a 96-well cell culture plate at a density of 1.2×104 cells per well and were allowed to adhere overnight in D -MEM (Gibco, Waltham, MA, USA) with 10% FBS (Thermo Scientific, Waltham, MA, USA). HEK cells were then co-transfected with 135 ng per well pMIR-Report Vector (with or without Sall4 3′ UTR insert or seed sequence mutagenized SAll4 3′ UTR insert) and 45 ng per well pMIR-Report beta gal vector (Life Technologies) with 100 nmol/l miR-219 mimic or mimic control using Lipofectamine 2000 (Invitrogen). Cells were incubated for 48 h and luminescence was detected using the Dual Light System (Ambion) using the manufacturer’s protocol.
6
Androgen Receptor Transactivation Assay
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For AR transactivation studies, COS-1 or HeLa cells were seeded into 12-well tissue culture plates in Dulbecco's modified essential medium (DMEM) containing 2 mM glutamine and 10% charcoal-stripped serum. Cells were transfected with 250 ng pGRE2-TATA-Luc, 25 ng pSVAR0 and a control reporter 25 ng phRG-TK (Renilla Luciferase) or pCH110 (β-galactosidase) as indicated using standard transfection procedures. After 16 h incubation the cells were exposed to fresh medium containing 0–10 nmol dihydrotestosterone (DHT; Sigma) or the synthetic androgen mibolerone (Steraloids Inc) for a further 24 h. The cells were then lysed in 500 μl passive lysis buffer (Promega) and the ratio of firefly to renilla was determined using Nanolight technology Alternatively, Dual-light System (Applied Biosystems) was used to measure Luciferase and β-galactosidase activities. Reporter assays were quantified using a Microplate Luminometer LB 960 (Berthold).
7
Luciferase Assay of NF-κB Reporters
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The DNA constructs of pNF-68-Luc and pNF-200-Luc reporter genes in a pLightSwitch_Prom vector were purchased from Active Motif. Cultured PC 12 cells were transiently transfected with pNF-68-Luc and pNF-200-Luc by lipofectamine 3000 reagent (Invitrogen) [13 (link)]. The transfected cells were exposed to the NGF and CM treatment and a luciferase assay was performed using a Dual-light® System (Applied Biosystems, CA, USA) with K252a as a positive control. The transfected cells were lysed with phosphate lysis solution at pH 7.8 with 0.2 % triton and centrifuged at 16,000×g at 4 °C for 10 min (Eppendorf, Hamburg, Germany). The cell lysates were transferred to 96-well assay plate and set on the GloMax™ 96 Microplate Luminometer (Promega, UK). The intensities of each sample were normalized using protein concentration.
8
Dual-Light System Reporter Assay
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Reporter gene assays were performed using the Dual-Light System (Applied Biosystems). ∼50% confluent HEK-293 T were transfected with SMARTpool siRNAs (12 pmoles cm−2) in six-well cell culture plates (Falcon) using jetPRIME reagent (Polyplus Transfection) following the manufacturer's instructions. Twenty-four hours after siRNA transfection, TEAD reporter plasmid (8xGTIIC–LUX56 (link); a gift from Stefano Piccolo, University of Padova, Italy; Addgene #34615) was transfected into the cells (200 ng cm−2) together with β-galactosidase plasmid (pEF-1α-LacZ 200 ng cm−2, Addgene #17430; kindly provided by Dr Shukry Habib, CSCRM, King's College London, UK) to normalize for transfection efficiency, using jetPRIME reagent (Polyplus Transfection) following the manufacturer's instructions. At 24 h post-plasmid transfection, cells were trypsinized and re-plated (1 × 104 cells) in triplicates into a 96-well plate, and allowed to grow for a further 24 h. Relative luciferase units were measured on a Glo-Max-Multi+ Multimode Reader (Promega) and normalized against β-galactosidase activity.
9
Dual-reporter splicing assay in HEK293
10
Validating miR-467b-3p regulation of Gpat1
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The pMIR-REPORT System (Thermo Fisher Scientific) was used to identify miR-467b-3p binding sites in the 3ʹUTR of Gpat1. The β-galactosidase reporter control vector was used for normalization. The putative or mutated miR-467b-3p binding sites of Gpat1 3ʹUTR were cloned into a luciferase miRNA expression reporter vector ,which contains firefly luciferase under the control of a mammalian promoter/terminator system. Hepa1–6 cells were co-transfected with the Gpat1 3ʹUTR luciferase reporter and the control construct, and with the miR-467b-3p mimic or mimic control, using the Lipofectamine 2000 system (Thermo Fisher Scientific). After 48 hours of transfection, luciferase activity was measured using the Dual-Light System (Applied Biosystems) and normalized to the corresponding β-galactosidase activity.
For the measurement of HNF4α signaling, Hepa1–6 cells were transfected with negative control or inducible HNF4α reporter plasmid from the Cignal HNF4α Reporter Assay Kit (SABiosciences, Hilden, Germany), along with a constitutively expressed Renilla construct. After 24 hours of transfection, cells were treated with 6-G for 48 hours. Luciferase activity was measured using the Dual-Light System.
For the measurement of HNF4α signaling, Hepa1–6 cells were transfected with negative control or inducible HNF4α reporter plasmid from the Cignal HNF4α Reporter Assay Kit (SABiosciences, Hilden, Germany), along with a constitutively expressed Renilla construct. After 24 hours of transfection, cells were treated with 6-G for 48 hours. Luciferase activity was measured using the Dual-Light System.
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