The HEK293T cells (American Type Culture Collection, Manassas, VA, United States) at the logarithmic phase of growth were seeded in 6-well plates at a density of 2 × 105 cells/well. After cell adherence, transfection was carried out according to the aforementioned method. After successful transfection, cells were cultured for 48 h and harvested. The SDC2-3’UTR-wild type (wt) and SDC2-3’UTR-mutant (mut) sequences were cloned into pGL3 vectors via restriction endonuclease, and then co-transfected with mimic NC or miR-9 mimic into HEK293T cells. Lipofectamine 2000 reagents (Invitrogen, Carlsbad, California, United States) were used for cell transfection. The luciferase activity was determined using the Dual-Luciferase Reporter Gene Assay kits (D0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions on a Glomax20/20 Luminometer (E5311, Shanxi Zhongmei Biotechnology Co., Ltd., Xi’an, China). The relative luciferase activity was calculated as ratio of firefly luciferase activity/renilla luciferase activity.
Dual luciferase reporter gene assay kit
Manufactured by Solarbio
Sourced in China
The Dual Luciferase Reporter Gene Assay Kit is a laboratory equipment used to measure the activity of two different luciferase reporter genes simultaneously. It provides a rapid and quantitative method for analyzing gene expression and transcriptional regulation.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ripa reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffer solution (pbs), by Thermo Fisher Scientific (1 mentions)
Qrt pcr and reverse transcription kits, by Transgene (1 mentions)
Multifunctional microplate reader, by Agilent Technologies (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Glomax20 20 luminometer fluorescence detector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pmirglo vector, by Promega (1 mentions)
9 protocols using dual luciferase reporter gene assay kit
1
Examining miR-9 Regulation of SDC2 3'UTR
2
CCK-8 Assay and Transwell Invasion in Cell Lines
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CCK-8 assay kits were purchased from Bestbio(BB-4202). Transwell kits were purchased from BioGenius, Shanghai (Transwell). qRT-PCR and reverse transcription kits were purchased from TransGen Biotech, Beijing, China (AQ141-01, AQ141-01). DMEM, phosphate buffer solution (PBS), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco, USA (1,142,802, 10,566,024, 10,010,023, 26,400,044, 15,070,063). RIPA reagents, BCA protein assay kits, ECL luminescence reagents, trypsin, and Lipofectamine™ 2000 Transfection Reagent were purchased from Thermo Scientific, USA (89,900, 23,250, 32,209, 90,059, 11,668,019). IGF-1 was purchased from Shanghai Hengfei Biotechnology Co., Ltd. (K002504P). β-actin primary antibody and goat anti-mouse IgG secondary antibody that was labeled by horseradish peroxidase (HRP) were purchased from R&D, USA (MAB8929, HAF007). Annexin V/PI apoptosis detection kits were purchased from Yisheng Biotechnology Co., Ltd., Shanghai, China (40302ES20). Dual luciferase reporter gene assay kits were purchased from Solarbio, Beijing, China (D0010). A PCR instrument was purchased from ABI, USA (7500). A flow cytometer was purchased from BD, USA (FACS Canto II). A multifunctional microplate reader was purchased from BioTek, USA (DLK0001622). All primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.
3
Quantifying IRAK1 Luciferase Activity
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Cells were seeded into the 6-well plates at a density of 2 × 105 cells/well. After cell adherence, cells were transfected according to the above methods. After successful transfection, cells were cultured for 48 h and collected. The luciferase activity of IRAK1 was detected according to the instructions of Dual Luciferase Reporter Gene Assay Kit (D0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The luciferase intensity of IRAK1 was detected using Glomax20/20 luminometer fluorescence detector (E5311, Shanxi Zhongmei Biotechnology Co., Ltd., Xi’an, China).
4
Dual-Luciferase Reporter Assay for LINC00472 and KLLN
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The WT and mut reporter plasmids of LINC00472 (WT-LINC00472 and mut-LINC00472) and the WT and mut reporter plasmids of KLLN (WT-KLLN and mut-KLLN) were provided by Shanghai GenePharma (Shanghai, China). The NC mimic, miR-149-3p mimic, and miR-4270 mimic were respectively co-transfected with WT-LINC00472, mut-LINC00472, WT-KLLN, and mut-KLLN into 293T cells. The cells were cultured for 48 h, with the changes in luciferase activity measured based on the instructions of the dual-luciferase reporter gene assay kit (D0010, Beijing Solarbio Technology, Beijing, China). The fluorescence intensity was detected using GLomax 20/20 Luminometer (E5311, Shaanxi Zhongmei Biotechnology, Xi’an, Shaanxi, China). The experiment was repeated three times.
5
Dual Luciferase Assay to Validate miRNA Targets
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A Dual Luciferase Reporter Gene Assay kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to assess the target genes of miR-378a-3p (Table I ). Cells were co-transfected with the wild-type/mutant plasmid DNA of the respective firefly luciferase target genes + Renilla luciferase plasmid DNA ± miR-378a-3p plasmid DNA, and cultured for 24/48 h. Lysate was collected after cells (200 µl lysate/1×106 cells) were fully lysed at 4°C for 5 min. A total of 20 µl cell lysate was added to 100 µl 1X firefly/Renilla luciferase reaction solution, and luciferase activity was detected. Firefly luciferase activity/Renilla luciferase activity was measured as luciferase reporter gene activity.
6
Stat3 3'UTR Luciferase Assay
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Stat3 3’UTR sequence containing miR-15a-5p binding sites was inserted into the pmirGLO vector to construct Stat3-wild type (Stat3-WT). Meanwhile, Stat3-mutant type (Stat3-MT) plasmid containing the target sites was also constructed. The above plasmids were transfected into KGN cells with miR-15a-5p or mimic-NC using Lipofectamine 3000 (Invitrogen). Based on the manufacturer’s protocols, the luciferase activity was examined using a dual-luciferase reporter gene assay kit (Solarbio, Beijing, China).
7
CDX2 and miR-181d-5p Binding Assay
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The target relationship, as well as the binding sites between CDX2 and miR-181d-5p, was analyzed, based on biological prediction website https://cm.jefferson.edu/rna22/Interactive/ , which was verified by dual luciferase reporter assay. The 3′ UTR region of CDX2 containing miR-181d-5p binding sites (CDX2-WT) and the mutant form in which the binding sites were mutated (CDX2-MUT) were inserted into the luciferase reporter vector. 293T cells were transfected with CDX2-WT or CDX2-MUT, together with miR-181d-5p mimic. 48 h later, the luciferase activity was measured following the manuals of the dual luciferase reporter gene assay kit (D0010; Beijing Solarbio Life Sciences, Beijing, China) by the GloMax 20/20 Luminometer fluorescence detector (Promega, Madison, WI, USA).
8
Dual-Luciferase Assay for Cell Lysis
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According to the instructions, the lysis cells were processed by the Dual-Luciferase Reporter Gene Assay Kit (D0010-100 T, Solarbio, CHN), and the relative light unit was measured at 560 and 465 nm with the microplate reader.
9
Validating miR-136-5p Binding Sites
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3ʹUTRs of circ_RANBP9 and XIAP containing miR-136-5p binding sites were inserted into pmirGLO vectors (Promega, Madison, WI, USA). Plasmids with the target-site mutations were constructed (MUT circ_RANBP9 and MUT XIAP). HEK293T cells were seeded in 24-well plates. The cells were co-transfected with wild type (WT) plasmids or the corresponding MUT plasmids and miR-136-5p mimic or mimic NC using Lipofectamine 2000 (Invitrogen). Luciferase activity was analyzed using the Dual Luciferase Reporter Gene Assay Kit (Solarbio, Beijing, China) following the manufacturer’s instructions.
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