Transcription factor-binding sites in the promoter region of human miR-551b were predicted by TargetScan and PicTar4 biological analysis website: http://www.targetscan.org and http://www.pictar.org . The putative ERBB4-binding site was 5′-AUGGGUCGA-3′, and the mutant ERBB4-binding site was 5′-UGUUUGAU-3′. The ERBB4 luciferase reporter constructs were made by amplifying the human ERBB4 mRNA 3′-UTR sequence by PCR and then cloning it into the XbaI site of the pGL3-promoter construct. The sequences of the human miR-551b promoter region were inserted into pGL3-basic vector (Sangon Biotech, Shanghai, China). The plasmids were then co-transfected with Renilla luciferase expression vector (pRL-TK) into MNK45 cells by Lipofectamine 2000 (Beyotime, Hangzhou, China) according to the manufacturer's instructions. After transfection for 24h, the luciferase activity was measured in samples by Dual-Luciferase Assay Kit (Beyotime, Hangzhou, China).
Dual luciferase assay kit
Manufactured by Beyotime
Sourced in China, United States
The Dual-luciferase assay kit is a laboratory instrument designed to measure the activity of two luciferase reporter enzymes simultaneously. It provides a rapid and sensitive method for quantifying gene expression levels in experimental samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (15 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Psicheck 2 vector, by Promega (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 6000, by Beyotime (2 mentions)
Prl tk, by Promega (2 mentions)
Psicheck 2, by Promega (2 mentions)
Lipofectamine 2000, by Beyotime (1 mentions)
Pgl3 basic vector, by Sangon (1 mentions)
Alamar blue assay, by Solarbio (1 mentions)
Spectramax m5 microplate reader, by Molecular Devices (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Mithras lb940 multilabel reader, by Berthold Technologies (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Pgl3 basic reporter plasmid, by Promega (1 mentions)
Tca8113, by Procell (1 mentions)
Cal 27, by Procell (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
51 protocols using dual luciferase assay kit
1
miR-551b Transcriptional Regulation by ERBB4
2
Regulation of CCND2 and AKT3 by miR-610
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CCND2 and AKT3 were regulated by miR-610 through direct binding to its 3′-UTR, a series of 3′UTR fragments including the full-length wild-type 3′-UTR, binding site mutants were constructed and inserted into the psiCHECK2 luciferase reporter plasmid (Invitrogen Life Technologies). Cells were seeded in triplicate in 24-well plates (5×104/well) and cultured for 24 h. The pGL3-luciferase reporter gene plasmids pGL3-AKT3-3′-UTR (wild or mut) or pGL3-CCN2-3′-UTR (wild or mut) and the relative control-luciferase plasmid were cotransfected into the KEK293T cells with the relative control pRL-TK Renilla plasmid (Promega Corportation) using Lipofectamine 2000 reagent (Invitrogen Life Technologies). Luciferase and Renilla activities were assayed 48 h after transfection, the cells were lysed and the fluorescence intensity was detected using the dual luciferase assay kit (Beyotime Institute of Biotechnology, Haimen, China).
3
Validation of miR-200b-3p and miR-429-5p Regulation of LIMK1 3'UTR
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HEK 293T cells were seeded in 48-well plates and cultured until they reached 70% confluence. The psiCHECK-2/LIMK1 3′-untranslated region (UTR) and psiCHECK-2/LIMK1 3′-UTR mutant reporter plasmids were purchased from Ibsbio. HEK 293T cells were transiently cotransfected with 0.2 µg psiCHECK-2/LIMK1 3′-UTR or psiCHECK-2/LIMK1 3′-UTR mutant reporter plasmids together with 100 nM/L miR-200b-3p mimics, miR-429-5p mimics, or NC mimics using Lipofectamine 2000 according to the manufacturer’s instructions. After 72 h, cell lysates were collected to determine firefly and Renilla luciferase activity using a Dual Luciferase Assay Kit (Beyotime Biotechnology). Firefly luciferase activity values were normalized to the values for Renilla luciferase, and the results are presented as the ratio of firefly to Renilla activity values.
4
NLRP3 3'UTR Structural Analysis and Luciferase Assay
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A 439-bp mRNA in the 3′UTR of NLRP3 containing rs10754558 G-to-C mutant was submitted to the RNAfold Web Server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ) setting default parameters to produce positional entropies, centroid structures and Potential minimum free energy (MFE) structures. Next, a fragment with 313 bp length in the 3′UTR region of NLRP3 containing rs10754558 G-to-C mutant was inserted downstream to luciferase reporter gene in pmirGLO luciferase reporter vectors. Lipofectamine 3000 (Invitrogen, MD, USA) was used for cell transfection, and dual-luciferase assay kit (Beyotime, Shanghai, China) was used to measure the activities of luciferase in a SpectraMax L Chemiluminescent reader (Molecular Devices, USA).
5
E-cadherin Promoter Activity Assay
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LoVo cells (1 × 105) were firstly seeded in 6-well plates and incubated overnight in a humidified, 5% CO2 atmosphere, at 37°C. To test E-cadherin (CDH1) promoter activity, LoVo cells were cotransfected with either the recombinant plasmid pGL3-basic-CDH1-promoter or pGL3-basic-mut-CDH1-promoter, with a control positive plasmid pRL-SV40. After transfection for 24 h, the promoter activity was measured using a dual-luciferase assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions.
6
Dual-Luciferase Assay for circTLK1 and Smad3
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Both wild and mutant-type vectors were constructed in GenePharma company. In brief, MUG-Chor1 cells transfected with wild and mutant-type circTLK1 or Smad3 were lysed followed by a dual-luciferase assay kit (Beyotime Biotechnology, Cat# RG027). Finally, a microplate reader (Bio-Tek, Santa Clara, CA, USA, Model No. SLXFA) was used to detect relative luciferase intensity.
7
Examining c-Myc Impact on RAP2 Expression
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To examine the impact of c-Myc on RAP2 expression, the pGL3-RAP2 vector was co-transfected with the c-Myc and Renilla luciferase vectors into PANC-1 and SW1990 cells. Luciferase activity was measured by using dual-luciferase assay kit (Beyotime Biotechno-logy, Shanghai, China) according to the manufacturers’ protocol.
8
Cloning and Characterizing PLVAP Promoter
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The DNA fragment from the human PLVAP promoter extending from −1727bp to −1bp, was cloned into the pGL3 Basic vector to generate the wild-type Luc construct. The mutant construct with deletion of the CRE site in the PLVAP promoter was generated. For luciferase assay, the luciferase reporter plasmid was transfected into HEK-293T cells in 24-well plates by using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, United States). The p-RL-TK plasmid carrying the Renilla luciferase under control of the thymidine kinase promoter was co-transfected as an internal control. After 24 h, cells were treated with forskolin and luciferase activity was analyzed using the Dual Luciferase Assay Kit (Beyotime, Nantong, China).
9
Dual Luciferase Assay in HEK293T and HEY Cells
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HEK293T and HEY cells were seeded in a 24-well plate (30,000 cells/well). After being transfected with the indicated plasmid and miRNA mimics, cells were harvested and subjected to luciferase reporter assay using the dual luciferase assay kit (Beyotime, Shanghai, China). The relative luciferase activity was calculated by dividing the firefly luciferase activity by the Renilla luciferase activity.
10
Characterizing circCASP9 Regulation by miRNAs
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The wild-type (WT) or mutant-type (MUT) plasmids (pGL3-Firefly_Luciferase-Renilla_Luciferase) containing circCASP9 and the KANK1 3′-UTR were designed and constructed by Gene Create (Wuhan, China). HEK293T cells were cotransfected with the plasmid and miR-589-5p mimic, miR-4664-3p mimic, or miR-NC using Lipofectamine 2000. After incubation for 48 h, a Dual-Luciferase Assay Kit (Beyotime, China) was applied to perform the luciferase reporter assay.
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