Vascular cell adhesion molecule 1 wild-type or mutant 3′UTR fragment was subcloned into pmirGLO (Promega, Madison, WI, United States). Until reaching 70% confluence, the cells were starved for 12 h and then co-transfected with 75 pmol miR-126-3p mimic or NC mimic and 1.5 μg recombinant plasmid containing wt or mut 3′UTR. Forty-eight hours post-transfection, luciferase activities were determined by Dual-luciferase reporter assay kit (KeyGen, China).
Dual luciferase reporter assay kit
Manufactured by Keygen Biotech
Sourced in China
The Dual-luciferase reporter assay kit is a laboratory tool used to measure and compare the activity of two different reporter genes simultaneously. It provides a standardized method for quantifying gene expression levels in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pmirglo, by Promega (1 mentions)
Cell lysis buffer, by Keygen Biotech (1 mentions)
Phosphate buffered saline (pbs), by Sangon (1 mentions)
Easy mutagenesis system kit, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Lipo8000 reagent, by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Lamin b, by Cell Signaling Technology (1 mentions)
Cecl western blot kit, by CoWin Biotech (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
Dimethyl sulphoxide dmso, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Prl sv40p, by Addgene (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
6 protocols using dual luciferase reporter assay kit
1
miR-126-3p Regulates VCAM-1 Expression
2
Evaluating SOX9-TXNIP Transcriptional Regulation
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For detection of the correlation between SOX9 and TXNIP predicted by the JASPAR website, Müller cells were co-transfected with the TXNIP promoter-reporter vectors and SOX9
overexpression vectors. After transfection for 48 h, the cells were washed twice with PBS (Sangon, Shanghai, China), and 250 µl of cell lysis buffer (KeyGen Biotech,
Nanjing, China) was then added. The luciferase activity was determined using the Dual-Luciferase Reporter Assay Kit (KeyGen BioTech) following the manufacturer’s protocol.
Renilla luciferase activity was used as an internal control.
overexpression vectors. After transfection for 48 h, the cells were washed twice with PBS (Sangon, Shanghai, China), and 250 µl of cell lysis buffer (KeyGen Biotech,
Nanjing, China) was then added. The luciferase activity was determined using the Dual-Luciferase Reporter Assay Kit (KeyGen BioTech) following the manufacturer’s protocol.
Renilla luciferase activity was used as an internal control.
3
Investigating miR-146a-3p Regulation of MBD2
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The wild-type (WT) MBD2 3′-untranslated region (3′-UTR) was amplified by PCR and cloned into the psiCHECK-2 vector (Promega, Madison, USA). MBD2 cloned to the psiCHECK-2 vector was mutated to obtain the mutant type (MT) MBD2 using the Easy Mutagenesis System Kit (Promega, Madison, USA). Then, 293-T cells were transfected with the WT-MBD2-3′-UTR or MT MBD2-3′-UTR plasmid (300 ng) and mimic miR-NC or mimic miR-146a-3p (100 nM), using Lipofectamine™ 3000 (Thermo Fisher Scientific) transfection reagent based on the manufacturer’s instructions. Forty-eight hours after transfection, a luciferase assay was performed using a dual-luciferase reporter assay kit according to the manufacturer’s instructions (Keygen biotech, Changsha, China).
4
FOXO3 Binding to CNN3 Promoter
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In order to confirm the binding between FOXO3 and the promoter sequence of CNN3, dual-luciferase reporter assay was executed in 293T cells. The cells were transfected with pGL3 vector (Shaanxi Youbio Technology Co., Ltd.) containing CNN3 promoter fragment and FOXO3 overexpression plasmid (Shaanxi Youbio Technology Co., Ltd.) at 37°C in the presence of Lipo8000 reagent (Beyotime Institute of Biotechnology) for 4–6 h. Following transfection for 48 h, the cells were collected and treated with a dual-luciferase reporter assay kit (Nanjing KeyGen Biotech. Co., Ltd.), and the Firefly and Renilla value was determined using a microplate reader (BioTek Instruments, Inc.).
5
Wnt Signaling Pathway Activation Assay
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Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F12 medium, trypsin, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from Gibco (CA, USA). Sulforhodamine B (SRB) and Dimethyl Sulphoxide (DMSO, purity>99.9%) were purchased from Sigma-Aldrich (MO, USA). Transwell systems were purchased from Corning (NY, USA). Matrigel basement membrane matrix was bought from BD Biosciences (NJ, USA). M50 Super 8x TOPFlash (12457), M51 Super 8x FOPFlash (TOPFlash mutant) (12457) and pRL-SV40P (27163) were obtain from Addgene. Lipofectamine 2000 was bought from Thermo (MA, USA). The dual luciferase reporter assay kit was obtained from KeyGEN Biotech (Jiangsu, China). The nuclear and cytoplasmic protein extraction kit was purchased from Beyotime Biotechnology (Jiangsu, China). Antibodies against Wnt-2, GSK-3β, c-myc, β-catenin, p-GSK-3β, Survivin, Lamin B and β-actin were purchased from Cell Signaling Technology (MA, USA). The cECL Western Blot Kit was obtained from CoWin Biotech (Beijing, China). All the chemical reagents were of the highest grade.
6
Wnt5a 3'UTR Regulation by miR-374
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Wild-type Wnt5a 3’UTR was cloned downstream of the pmirGLO (E133A,
Promega, Madison, WI, USA) vector. Mutations were performed in the binding sites. The
pmirGLO vector plus miR-374 agomir or negative control agomir were transiently
co-transfected into 293T cells (Zhong Qiao Xin Zhou Biotechnology, Shanghai, China). The
activities of firefly and Renilla luciferases were measured with a dual-luciferase
reporter assay kit (KeyGen Biotech, Nanjing, China).
Promega, Madison, WI, USA) vector. Mutations were performed in the binding sites. The
pmirGLO vector plus miR-374 agomir or negative control agomir were transiently
co-transfected into 293T cells (Zhong Qiao Xin Zhou Biotechnology, Shanghai, China). The
activities of firefly and Renilla luciferases were measured with a dual-luciferase
reporter assay kit (KeyGen Biotech, Nanjing, China).
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