We used the DIANA, RNA22, miRanda and miRWalk2.0 bioinformatics tools to predict the binding sites between lnc-PSMA8-1 and miR-144-3p and between the mTOR 3’UTR and miR-144-3p. Luciferase plasmids that contained the wild-type lnc-PSMA8-1 binding site (lnc-PSMA8-1-WT) or mutated lnc-PSMA8-1 binding site (lnc-PSMA8-1-MUT) or the wild-type mTOR 3’UTR (mTOR 3’UTR-WT), or mutated mTOR 3’UTR (mTOR 3’UTR-MUT), the corresponding empty vector controls, and the Renilla luciferase plasmid were constructed by Shanghai GeneChem Co., Ltd. RD and RH30 cells were seeded into 24-well plates (3 × 104 cells/well); 24 h later, the cells were transfected with 0.1 µl of one of the luciferase plasmids, 0.02 µl of the Renilla luciferase expression plasmid, and 100 nM miR-NC or the miR-144-3p-mimic. After 48 h, Renilla luciferase expression was measured according to the manufacturer’s protocol.
Renilla luciferase plasmid
Manufactured by Genechem
Sourced in China
The Renilla luciferase plasmid is a laboratory tool used for gene expression analysis. It encodes the Renilla luciferase enzyme, which catalyzes a bioluminescent reaction that can be detected and measured. The plasmid serves as a reporter to monitor and quantify gene expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pmirglo luciferase vector, by Genechem (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Gv141 vector, by Genechem (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Epas1 cdna, by Genechem (1 mentions)
4 protocols using renilla luciferase plasmid
1
Characterization of lnc-PSMA8-1 and mTOR Regulation by miR-144-3p
2
Investigating miR-106a-5p Binding Sites
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The putative miR‐106a‐5p binding sites in lncPCAT1, E2F5, and BMP2, both wild‐type and mutant type, were synthesized and cloned downstream of the luciferase gene in pmirGLO luciferase vectors (Genechem). The plasmids, miRNA mimics (miR‐NC and miR‐106a‐5p), miRNA inhibitor (control anti‐miRNA, anti‐ miR‐106a‐5p) and Renilla luciferase plasmid (Genechem), used as a normalization control, were cotransfected into cells using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were then measured consecutively by Dual Luciferase Assay 48 hr after transfection. All experiments were repeated three times.
3
Transcriptional Regulation by p53 and SMAD3
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Plasmids encoding p53 in the GV141 vector were purchased from GeneChem (Shanghai, China). Both WT and mutant SMAD3 genes were synthesized by chemical methods and cloned downstream of the luciferase gene in GV238 luciferase vectors that were obtained from GeneChem (Shanghai, China). These plasmids and a Renilla luciferase plasmid (GeneChem, Shanghai, China), which was used as a normalization control, were cotransfected into cells using Lipofectamine 3000 (Invitrogen, Shanghai, China). 293T cells (8 × 103 per well) were seeded in 96-well plates the day before transfection, and the cells were cotransfected with 1.25 μg of GV238-SMAD3 and 0.75 μg of p53 DNA when they reached 70%–90% confluence. In total, 50 ng of the expression plasmid (Renilla) was cotransfected as a transfection efficiency control. After 24 h, the cells were harvested, washed three times with PBS, and lysed in 20 μL of passive lysis buffer (PLB) according to the manufacturer’s instructions (Promega, Madison, WI, USA). Cell debris was removed by centrifugation, and the supernatant was used for a luciferase assay read with SpectraMax single-mode microplate readers (Molecular Devices, Sunnyvale, CA, USA).
4
Transcriptional Regulation of BCRP
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293T or OVCAR‐3 cells (1 × 104 per well) were seeded in triplicate in 96‐well plates. After 24 h, cells were transiently transfected with either 0.1 μg GV238‐basic vectors containing upstream promoter regions of BCRP (GV238‐BCRP‐WT), vectors containing mutated HRE sequence binding sites (GV238‐BCRP‐mut), or NC promoter plasmids and 0.01 μg Renilla luciferase plasmid and 0.2 μg EPAS1‐cDNA or NC‐cDNA plasmids (all from Shanghai Genechem Co., Ltd) using Lipofectamine 3000 transfection regent. Luciferase and Renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.
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