Details of these experiments are given in the Supplementary material . In short, a 52 base pair putative miR-206 target sequence from the 3′ UTR region of LXR, or a scrambled negative control was cloned using PremiR-Reporter constructs and synthetic Pre-miR206 from Ambion and the expression plasmid for miR206 from SBI (System Biosciences, PA, USA).
For the luciferase assay, 100 ng/ml of the PremiR-Reporter construct was co-transfected with 25 ng/ml β-Gal control plasmid and also with the 100 nM synthetic precursor-miR206 strand or the 1 μg/ml of miR-206 expression vector into COS-7 cells using X-tremeGENE siRNA Transfection Kit (Roche). After 36 h, cells were lysed and 50 μl of the lysate was used for β-Gal assay and 10 μl (2×) was used for luciferase assay carried out using the manufacturer’s protocol.
For the luciferase assay, 100 ng/ml of the PremiR-Reporter construct was co-transfected with 25 ng/ml β-Gal control plasmid and also with the 100 nM synthetic precursor-miR206 strand or the 1 μg/ml of miR-206 expression vector into COS-7 cells using X-tremeGENE siRNA Transfection Kit (Roche). After 36 h, cells were lysed and 50 μl of the lysate was used for β-Gal assay and 10 μl (2×) was used for luciferase assay carried out using the manufacturer’s protocol.