For dual-luciferase assay, AKT2 3’-UTR containing predicted let-7a seed-matching sites (wide type, WT) and corresponding mutant sites (mut) were amplified by PCR from cDNA of TPC-1 and inserted into pMIR-REPORTER vector (Ambion, CA, USA). Wild-type and mutant constructs were confirmed by sequencing. 293Tcells were seeded in a 24-well plate and cotransfected with either wild-type (WT) or mutant-type (mut) luciferase reporter plasmids containing AKT2-3’-UTR, pGL4.74 vector (Ambion), and equal amounts of let-7a, miR-NC (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen) according to the manufacturer′s instruction. Luciferase activities were measured 24 h after transfection using a dual luciferase assay kit (Promega, WI, USA). Experiments were performed in triplicate with three independent replicates.
Pgl4.74 vector
Manufactured by Thermo Fisher Scientific
Sourced in China
The PGL4.74 vector is a plasmid-based expression vector designed for use in various research applications. It serves as a backbone for cloning and expressing genes of interest in mammalian cell lines. The vector contains the necessary elements for DNA replication, selection, and gene expression, including a multiple cloning site and a reporter gene.
Automatically generated - may contain errors
2 protocols using pgl4.74 vector
1
Dual-Luciferase Assay for AKT2 3'-UTR
2
Dual-Luciferase Assay for miR-34a Targeting Sox7
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For dual-luciferase assays, the Sox7 3′ UTR containing predicted mir-34a seed-matching sites (wild-type; WT) and corresponding mutant sites (mut) were amplified by PCR from cDNA of GH4C1 and inserted into the pMIR-REPORTER vector (Ambion; Austin, TX, USA). WT and mutant constructs were confirmed by sequencing. GH4C1 cells were seeded in a 24-well plate and cotransfected with either WT or mutant luciferase reporter plasmids containing the Sox7-3′ UTR, pGL4.74 vector (Ambion), and equal amounts of mir-34a and miR-NC (GenePharma, Shanghai, China), using Lipofectamine 2000 according to the manufacturer’s instructions. Luciferase activities were measured 24 hr after transfection using a dual luciferase assay kit (Promega, Madison, WI, USA). Experiments were performed in triplicate with three independent replicates.
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