MicroRNA Target Validation Using Luciferase Reporter Assay

The biological target of the miRNA was predicted by scanning the seed region between the bta-miR-34b seed region and the predicted gene on TargetScan (http://www.targetscan.org). The mature sequence of bta-miR-34b was obtained from the miRBase Sequence Database (http://www.mirbase.org). The sequence and the 3′ untranslated region (UTR) mRNA of DCP1A from Bos taurus were aligned via the University of California-Santa Cruz Genome Bioinformatics program (http://genome.ucsc.edu). The DCP1A-3′ UTR wild-type (WT) and mutation type (MUT) were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI) using the XhoI and NotI restriction sites. 293T cells were cultured in 96-well plates and transfected when cells were grown to 70% confluence. Approximately 0.16 µg of DCP1A-3′ UTR WT or MUT vector was cotransfected with 5 pmol miR-34b mimics or a negative control into 293T cells using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA). After 48 h of transfection, luciferase activity was measured using the luciferase reporter assay system (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to the activity of Renilla luciferase.