Dual-Luciferase Assay for VEGF 3'UTR

Rat VEGF 3′-UTR (nt1660–3545) was inserted into the Dual-Luciferase reporter vector (pEZX-MT01, Genecopoeia Corp. MD, USA) downstream from the Firefly luciferase (hLuc) reporter gene, and was driven by SV40 Enhancer promoter. In addition, Renilla luciferase (hRLuc) reporter driven by a CMV promoter was cloned into the same vector, serving as the tracking gene and internal control. The dual-reporter vector system enabled transfection-normalization for accurate across-sample comparison. The 293TN cells were assigned into three groups to be transfected with A. pEZX-MT01 vector; B. pEZX-MT01 vector+NC-miR-377 mimic; C. pEZX-MT01 vector+miR-377 mimic. Cell lysates were collected and assayed 48 hours after transfection. Firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System kit (Promega Corp. WI, USA) and each transfected well was assayed in triplicate as described [27] (link). The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs. MA, USA) with the following primer, mutated VEGF 3′UTR forward primer 5′-AAGGATAAAATAGACATTGCTATTCTG-3′; reverse primer 5′-AGACTATATACATAAACATATATATATATATATACAC-3′.