The 3’UTRs of CACNB3 (1116 bp, refseq. NM_000725.3), DDN (1698 bp, refseq. NM_015086.1) and KLC2 (964 bp, refseq. NM_001134775.1) were PCR amplified from human brain cDNA (Invitrogen) and cloned downstream of the phosphoglycerate kinase I (PGK) promoter driven the luciferase reporter gene luc2 expression in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI). The 3’UTR of ANK3 (refseq. NM_001149.3), CACNA1C (refseq. NM_199460.2) and SYT1 (refseq. NM_005639) were cloned by GeneCopoeia downstream of the SV40 promoter driven the secreted Gaussia luciferase (GLuc) reporter gene in a firefly/Renilla Duo-Luciferase reporter vector (pEZX-MT01). For miR-34a target validation, HEK293T cells were seeded at a density of 104 cells per well in a 96-well plate and grown for 24h before transient transfection with 20–40 ng of each dual luciferase reporter construct and 30 nM of pre-miR-34a or pre-miR-Neg (Ambion, Austin, TX) using Lipofectamine 2000 (Invitrogen). Luciferase activities were measured 48h after transfection using the Dual Glo Luciferase Assay System (#E2920, Promega) or the Luc-Pair miR Luciferase Assay Kit (LPFR-M030, GeneCopoeia) depending on the luciferase reporter constructs. Efficiency of transfection was normalized using Renilla luciferase activity.
