**2.9 Target validation using luciferase expression plasmids**

HepG2 cells were used for miR-665 target validation, because miR-665 does not inhibit the growth of these cells. When mouse neuroblastoma cells were used for target validation, the negative control luciferase vector plasmid without any target 3'-UTR showed a 50% decrease in luciferase activity when co-transfected with miR-665 compared to negative control miRNA. This decrease in luciferase activity was non-specific and was caused by cell growth inhibition due to miR-665 transfection.

To validate miR-665 targets, HepG2 cells were grown for 24 h in a 96-well plate. Cells were then co transfected with 100 ng luciferase expression plasmids containing the 3'-UTR for HDAC8, c-MYC, or MYCN, or the empty vector without any target 3'UTR, plus 100 nM negative control miRNA or miR-665 with Dharmafect Duo transfection agent. After 48 h of cotransfection, luciferase activity was measured using the Active Motifs LightSwitch luciferase assay kit. Luminescence was read on a Molecular Devices Soft Max Pro5 luminometer.
