293T cells were transfected with the Firefly luciferase plasmid (Promega Corporation) containing wild-type or mutant 3′-untranslated region (UTR) of Rap1b, using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.). The cells were subsequently transfected with NC, miR-101 mimic or anti-miR-101 (all from Ambion; Thermo Fisher Scientific, Inc.). After 48 h of cell culture, the cells were harvested and lysed. Luciferase activity was measured using the the Dual-Luciferase Reporter Analysis kit (Promega Corporation). Normalized relative light units represent Firefly luciferase activity/Renilla luciferase activity.
Dual luciferase reporter analysis kit
Manufactured by Promega
Sourced in United States
The Dual-Luciferase Reporter Analysis kit is a laboratory tool designed for the quantitative measurement of gene expression. It utilizes two distinct luciferase reporter enzymes to enable the simultaneous assessment of two different experimental conditions or promoters within the same sample.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Firefly luciferase plasmid, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Pmirglo reporter vector, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using dual luciferase reporter analysis kit
1
Rap1b 3'UTR Luciferase Assay
2
Circ_0000098 and ALX4 3'-UTR Luciferase Assay
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The wild type (WT) or mutant type (MUT) of circ_0000098 or ALX4 3’-UTR sequence was amplified and cloned into the psiCHECK-2 vector (Promega, Madison, WI, USA) to construct luciferase reporter plasmid (circ_0000098-WT/MUT or ALX4 3’-UTR-WT/MUT). The reporter plasmids, together with miR-1204 mimic or miR-NC, were co-transfected into Huh7 and SMMC-7721 cells by Lipofectamine™ 3000. After 48 h, the luciferase activity was detected by the dual-luciferase reporter analysis kit (Promega, Madison, WI, USA).
3
Validation of miR-524-5p binding to SChLAP1
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The wild-type (WT) or mutated (Mut) fragment of SChLAP1 containing the predicted binding sites of miR-524-5p was inserted into the pmirGLO reporter vector (Promega Corporation). TNBC cells were co-transfected with 50 nM miR-524-5p mimic and 200 ng WT or Mut luciferase plasmid of SChLAP1 using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The luciferase activity was measured after transfection for 48 h using the Dual-Luciferase reporter analysis kit (Promega Corporation) following the manufacturer's protocol. The activity of Renilla luciferase was detected to normalize the activity of firefly. The experiments were performed in three replicates.
4
Validating circRNA-miRNA Binding Interactions
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To confirm the binding between circRNA and miRNA, the HEK-293 T cells were co-transfected with psiCHECK2-hsa_circ_0098181-WT/-MUT (Promega, Madison, WI, United States) and miR-18a-3p NC/mimic (Ribobio, Guangzhou, China). Similarly, psiCHECK2 PPARA-3′UTR-576bp-WT/MUT1/MUT2/MUT1+MUT2 (MUT1 and MUT2 were single mutations) and miR-18a-3p NC/mimic were co-transfected in HEK-293 T cells to verify the combination between miR-18a-3p and PPARA. The cells were quantified using a dual-luciferase reporter analysis kit (Promega, Madison, WI, United States).
5
Regulation of Keap1 by miR-26b
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The downstream target genes of miR-26b were predicted using the TargetScan database (http://www.targetscan.org ). Keap1 was identified as a potential downstream target of miR-26b. 293T cells were obtained from the American Type Culture Collection and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO2. Luciferase reporter assays were used to investigate the regulatory relationship between miR-26b and Keap1. Site-directed mutagenesis was used to create the mutant 3′-untranslated region (UTR) sequence of Keap1. The wild-type and mutant-type sequences of Keap1 were cloned into the pGL-luciferase plasmid (Promega, Corp.), while the NC and miR-26b mimics sequences were co-transfected into 293T cells using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h incubation at 37°C, firefly and Renilla luciferase activities were evaluated using the Dual-Luciferase Reporter Analysis kit (Promega Corporation). Normalized relative light units represent firefly luciferase activity/Renilla luciferase activity.
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