pMIR-REPORT™ miRNA Expression Reporter Vector System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1×10^4 cells/well in 24-well plate	Transfect cells with 200 ng of the luciferase construct plasmid and 50 ng of pMIR-REPORT β-gal

Upstream tips
Seed 1×10^4 cells/well in 24-well plate
Protocol tips
Transfect cells with 200 ng of the luciferase construct plasmid and 50 ng of pMIR-REPORT β-gal

Publication protocol

Smad7 mRNA 3′-UTRs containing one miR-21-binding sequence for the mouse Smad7 gene (NCBI reference sequence: NM_001042660.1) were acquired by oligonucleotide synthesis following the manufacturer's instructions (pMIR-REPORT™ System, miRNA Expression Reporter Vector, Part No. AM5795, Ambion, Shanghai, China) (Table III). The annealing fragment was then subcloned into the SpeI site and HindIII site in the pMIR-REPORT™ miRNA expression reporter empty vector (Ambion, Shanghai, China). Binding-region mutations were achieved using an oligo synthesis following the manufacturer's instructions (Ambion). Transient transfection into HEK-293 cells (1×104 cells/well) was carried out in 24-well plates with Lipofectamine™ 2000 (Invitrogen, Shanghai, China) following the manufacturer's instructions. The cells were co-transfected with 200 ng of the luciferase construct plasmid and 50 ng of pMIR-REPORT β-gal (Ambion) plasmid, and luciferase assays were performed using the dual-luciferase reporter assay system (Ambion) according to the manufacturer's instructions. Luminescent signals were quantified using a luminometer (Promega, Madison, WI, USA), and each value from the firefly luciferase construct was normalized by β-gal assay.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for pMIR-REPORT™ miRNA Expression Reporter Vector System below.

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