Lightswitch 3 utr reporter goclone constructs of

The LightSwitch 3′-UTR reporter GoClone constructs of various putative miRNA targets were purchased from SwitchGear Genomics (Active Motif). The entire 3′-UTRs were placed downstream of a RenSP luciferase reporter under the control of the cytomegalovirus (CMV) promoter, thus the impact of a miRNA on regulation of transcript stability or translation efficiency could be assessed. For the assay, Huh7.5.1 cells were transfected with miRNA mimic of interest or with mimic control for 24 h in 96-well white plates (in five replicates), and then transfected with 50 ng of GoClone 3′-UTR reporter plasmid using FuGENE 6 transfection reagent (Roche). Two days later, cells were lysed and total luciferase outputs were read using LightSwitch Luciferase Assay Reagent (SwitchGear Genomics).

The LightSwitch 3′ UTR reporter GoClone constructs of various putative miR-135a targets were purchased from SwitchGear Genomics (Active Motif). Huh7.5.1 cells were transfected with miR-135a WT or MUT or with mimic control in 96-well white plates (in 5 replicates). After 24 h, cells were further transfected with 50 ng of GoClone 3′ UTR reporter plasmid using FuGENE 6 transfection reagent (Roche). Two days later, cells were lysed and total luciferase outputs were quantified using LightSwitch Luciferase Assay Reagent, according to the manufacturer’s instructions (SwitchGear Genomics) using a POLARstar multidetection microplate reader (BMG Labtech).