Lightswitch assay reagent

HepG2 cells were seeded in 96-well white and clear (to confirm the density of cells) TC plates in a total volume of 100 μL. The synthetic miRNA Target GoClone reporter hsa-miR-203 (SwitchGear Genomics, CA, USA) was used as a target vector, and EMPTY_3UTR, Empty vector (no 3′UTR) (SwitchGear Genomics) was used as a control vector. miCENTURY OX miNatural, has-miR-203 (Cosmo Bio, Tokyo, Japan) was used as the synthetic miR-203. miCENTURY OX miNatural, microRNA control, non-target RNA (Cosmo Bio) was used as the control. miNatural and miRCURY LNA microRNA Inhibitor, hsa-miR-203 (Exiqon, Vedbaek, Denmark) was used as an inhibitor of miR-203. miNatural and miRCURY LNA microRNA inhibitor, antisense control A (Exiqon) was used as the control. Individual vectors and microRNAs were mixed with serum-free media, and Lipofectamine 2000 reagent (Thermo Fisher Scientific) was diluted with Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific). The final concentrations of synthetic miR-203 controls were 0, 5, 10, 20, and 40 nM, and those of the inhibitor of miR-203 was 40 and 80 nM. The concentration of each control was 40 nM. One hundred microliters of the mixture were added to each well and incubated for 24 h. Luciferase activity was measured using LightSwitch Assay Reagents (SwitchGear Genomics) and Luminescencer JNR IKB2300 (ATTO, Tokyo, Japan) according to the manufacturer’s protocol.

Confirmation of miR-149-binding to the 3′ UTR of ABCC3 and of miR-378 and miR-422-binding to the 3′ UTR of TMEM45B. HEK 293 cells at 80% confluency were co-transfected with luciferase reporter plasmids harboring the complete 3′-UTR of the desired gene (SwitchGear Genomics) along with 100 nM of each miR-mimic or miRNA control (Sigma). DharmaFECT Duo (Thermo Scientific) was used as the transfection reagent in Opti-MEM (Life Technologies). Luminescence was assayed 24 hours later using LightSwitch Assay Reagents (SwitchGear Genomics) according to the manufacturer’s instructions. Knockdown was assessed by calculating luciferase signal ratios for specific miRNA/non-targeting control, using empty reporter vector as control for non-specific effects. Each experiment was performed in triplicate. t -test was performed for wells from multiple experiments, and we compared mimic-transfected cells with a mimic control for each gene vector.

Confirmation of miR-107-binding to the 3′-UTR of CCDN1. HEK 293 cells at 80% confluency were co-transfected with luciferase reporter plasmids harboring the complete 3′-UTR of the desired gene (SwitchGear Genomics) along with 100nM of miR107-mimic or miRNA control (Sigma). DharmaFECT Duo (Thermo Scientific) was used as the transfection reagent in Opti-MEM (Life Technologies). Luminescence was assayed 24 hours later using LightSwitch Assay Reagents (SwitchGear Genomics) according to the manufacturer's instructions. Knockdown was assessed by calculating luciferase signal ratios for specific miRNA/non-targeting control, using empty reporter vector as control for non-specific effects. Each experiment was performed in triplicate