Dual-luciferase reporter assays were conducted using a Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's instructions. Brie y, UBE2T-Wt/Mut were constructed using the psi-CHECK 2 plasmid (Promega, USA) by restriction endonuclease digestion and T4 DNA ligase, then they were co-transfected with miR-182-5p mimics/miR-NC into ccRCC cells using Lipofectamine 3000 (Invitrogen, USA) and incubated for 48h. Finally, the luciferase activities were measured on a microplate reader by using a luciferase assay kit (KeyGEN BioTECH, China).
Luciferase assay kit
Manufactured by Keygen Biotech
Sourced in China
The Luciferase assay kit is a laboratory tool used to measure the activity of the luciferase enzyme. Luciferase is a bioluminescent protein that emits light when catalyzing the oxidation of its substrate, luciferin. The assay kit provides the necessary reagents and protocols to quantify luciferase activity in cell lysates or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
M200 pro, by Tecan (2 mentions)
Luciferase assay reagent, by Tecan (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pglo vector, by GenScript (1 mentions)
5 protocols using luciferase assay kit
1
Dual-Luciferase Assay for miRNA Binding
2
Analyzing Nr4a1 Binding Sites in HPGD Regulation
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There were ve Nr4a1 binding sites predicted in the upstream regions of transcriptional start sites of HPGD. B1: CCTGTCCTTTCT (-1920 ~-1909); B2: CTTGACTTTTGG (-1674 ~ -1663); B3: ATAAATGCCAAT (-948 ~ -937); B4: AAAACGTCAG (-554 ~ -545); and B5: GCAAAGATCACC (-176 ~ -165). According to their distribution, we set up ve groups to perform dual-luciferase assay. The rst group included these 5 regions, the second group included the other four motifs of B1, the third group included the three motifs B3, B4, and B5, the fourth group included the two motifs B4 and B5, and the fth group only contained the B5 motif. After transfection of plasmids containing above regions, cells were washed twice with PBS. Cell lysate was mixed with Luciferase Assay Reagent and then detected by a microplate reader (M200Pro, Tecan, Switzerland). The luciferase assay kit was purchased from KeyGEN BioTech (China).
3
Dual-Luciferase Assay for miRNA Binding
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Dual-luciferase reporter assays were conducted using a Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's instructions. Brie y, UBE2T-Wt/Mut were constructed using the psi-CHECK 2 plasmid (Promega, USA) by restriction endonuclease digestion and T4 DNA ligase, then they were co-transfected with miR-182-5p mimics/miR-NC into ccRCC cells using Lipofectamine 3000 (Invitrogen, USA) and incubated for 48h. Finally, the luciferase activities were measured on a microplate reader by using a luciferase assay kit (KeyGEN BioTECH, China).
4
ITGBL1 Promoter Luciferase Assay
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The JDP2 overexpression or control vectors were transfected with the pGL3 vectors containing the candidate ITGBL1 promoter sequence (from -2000 bp to +50 bp or -1200 bp to +50 bp) into PANC-1 cells. After 24-h transfection, the cells were examined for the luciferase activity using the luciferase assay kit (KeyGen Biotech). The enzyme-labeled instrument M200Pro (TECAN, Switzerland) was used to investigate the luciferase activity of the ITGBL1 promoter.
5
Regulation of PHF19 by LINC_00355 and miR-15a-5p
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The binding sites of LINC_00355 and miR-15a-5p were predicted by bioinformatics analysis. (LncBase v.2, http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted ). The binding sites of miR-15a-5p and PHF19 were predicted by TargetScanHuman 7.2 (http://www.targetscan.org/vert_72/ ). The wild type (wt) and mutant (mut) fragments of LINC_00355 were cloned into pGLO vector (GenScript, Nanjing, China) to construct LINC_00355-wt and LINC_00355-mut plasmids. The 3′-untranslated region (3′-UTR) sequences of PHF19 containing predicated or mutated miR-15a-5p binding sites were used to construct PHF19 3′-UTR-wt and PHF19 3′-UTR-mut plasmids. Cells were co-transfected with miR-15a-5p mimic (12.5 or 25 pmol per well) or its NC (12.5 or 25 pmol per well) and corresponding reporter plasmids (0.5 μg per well) using Lipofectamine™ 2000 (Invitrogen). At 48 h after transfection, luciferase assay kit (KeyGEN, Jiangsu, China) was used to detect luciferase activity.
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