3' UTR sequences were obtained from public domain gene sequence databases and inserted downstream of a secreted Gaussia luciferase (GLuc) reporter gene, which is expressed under control of the SV40 promoter in mammalian cells. A chimeric mRNA is transcribed consisting of the GLuc and a 3' UTR target sequence. Thus, the study of mRNA-miRNA target interaction can be easily performed with a live cell assay for GLuc using only 10 µl of the cell culture medium. Besides using GLuc as the miRNA 3' UTR target reporter, a secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter is also cloned into the same vector (pEZX-MT05) and serves as the internal control. The dual-reporter vector system enables transfection normalization for accurate across-sample comparison.
In addition to the GLuc/SEAP dual-reporter vector system, 3' UTR sequences are also vailable in a Firefly/Renilla Duo-Luciferase reporter vector (pEZX-MT06).
For miRNA target validation using GeneCopoeia's pEZX-MT05 and pEZX-MT06 target clones, the Secrete-Pair™ Dual Luminescence Assay Kit and Luc-Pair™ Duo-Luciferase Assay Kit, respectively, are recommended.