To evaluate the effect of miR-139-5p on FGB expression, we transfected a plasmid containing predicted miRNA interaction sites (wild type and mutant) in hBMECs cells using liposomal amine RNAiMAX (Thermo Fisher Scientific). Two days after transfection, the luciferase reporter analysis system (Promega, USA) was used to measure Renilla luciferase activity by normalizing it to firefly luciferase activity.
Luciferase reporter analysis system
Manufactured by Promega
Sourced in United States
The Luciferase reporter analysis system is a laboratory equipment designed to measure and analyze luciferase reporter gene activity. The system provides a reliable and sensitive method for quantifying gene expression levels in various experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Pmirglo, by Promega (1 mentions)
Psicheck 2 dual luciferase vectors, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
5 protocols using luciferase reporter analysis system
1
Evaluating miR-139-5p Regulation of FGB
2
miR-223-5p Regulates ERG Expression
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Luciferase assay was performed according to previous report 9 (link). Brief, several microRNA targets gene prediction systems website were searched to find target genes of miR-223-5p (PicTar, microRNA, MiRBase and TargetSpy). We found that the 3′UTR of ERG mRNA fragment contained miR-223-5p responsive elements. It meant a potential target gene might be found. Then we performed a dual Luciferase® Reporter analysis system (Promega, E1960, USA) according to its instruction. We choose DU145 cells and co-transfected them with plasmid-Lipo2000 (Invitrogen, USA) complexes, which including miR inhibitor, miR mimic or miR negative control (NC) as well as ERG fragment.
3
Dual-Luciferase Assay for miRNA Targets
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For dual-luciferase assay on VPS9D1-AS1 and miRNA-30a-5p in this study, the amplified VPS9D1-AS1 mutant (VPS9D1-AS1-Mut) or wild-type (VPS9D1-AS1-Wt) 3′UTR was cloned into the multiple cloning sites of the downstream of pmirGLO (Promega, Madison, USA) luciferase reporter vector. Lipofectamine 2000 was used to co-transfect VPS9D1-AS1-Mut or VPS9D1-AS1-Wt vector with miRNA-30a-5p-mimic or NC-mimic into the cells.
For dual-luciferase assay on miRNA-30a-5p and KIF11, the amplified KIF1-Mut or KIF11-Wt 3’ UTR was cloned into multiple cloning sites of the downstream of pmirGLO (Promega, Madison, USA) luciferase reporter vector. Lipofectamine 2000 was utilized to co-transfect KIF11-Mut or KIF11-Wt vector with miRNA-30a-5p-mimic or NC-mimic vector into the cells. After 48 h of transfection, luciferase reporter analysis system (Promega) was introduced to assay firefly and renilla luciferase activities.
For dual-luciferase assay on miRNA-30a-5p and KIF11, the amplified KIF1-Mut or KIF11-Wt 3’ UTR was cloned into multiple cloning sites of the downstream of pmirGLO (Promega, Madison, USA) luciferase reporter vector. Lipofectamine 2000 was utilized to co-transfect KIF11-Mut or KIF11-Wt vector with miRNA-30a-5p-mimic or NC-mimic vector into the cells. After 48 h of transfection, luciferase reporter analysis system (Promega) was introduced to assay firefly and renilla luciferase activities.
4
Dual-Luciferase Assay for NRXN1 3'UTR
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For dual-luciferase detection, amplified mutant-type or wild-type NRXN1 3′-UTR (NRXN1-Mut or NRXN1-Wt) was cloned into the psiCHECK-2 dual-luciferase vectors (Promega, Madison, WI). Before transfection, 100 μl of TC cell FTC-133 (1.5 × 105 cells/ml) was inoculated into a 96-well plate, and then, NRXN1-Mut/NRXN1-Wt and miR-mimic/NC-mimic were cotransfected into FTC-133 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Forty-eight hours later, Firefly and Renilla luciferase activities were measured by the luciferase reporter analysis system (Promega, USA), and the experiment was repeated three times.
5
Wnt/β-catenin Signaling Reporter Assay
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As previously reported (27 (link)). ST2 cells were cultured into 24-well plates, and the Wnt/β-catenin signaling reporter TopFlash and FopFlash plasmids (Addgene, Cambridge, MA, USA) were cotransfected into ST2 cells using Lipofectamine 3000 (Invitrogen). The transfected ST2 cells were then treated with C91, L-ctrl, and Wnt3a conditioned media. At 72 hr post-transfection, luciferase analysis was performed using a luciferase reporter analysis system (Promega) according to the manufacturer’s instructions. Results are expressed as normalized TopFlash/FopFlash values, and results are expressed as the mean ± standard deviation (SD) of three replicates.
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