Luciferase reporter assay

As reported [7], the human LMX1A 3’-UTR with the putative binding sites of miR-9 (see the sites at [7]) was amplified by PCR and then inserted into the firefly luciferase reporter vector, pGL4.13 (luc2/SV40) (Promega), at the XbaI site and downstream from the stop codon of the luciferase gene. AGS cells were plated at 1 × 10⁵ cells per well into six-well plates (60% confluence), transfected with pGL4.13 construct along with the Renilla luciferase reporter vector and pRL-SV40 (Promega). Following the indicated genetic treatments, LMX1A 3’-UTR luciferase activity value was tested, and results were normalized to that in the control cells.