Glowmax navigator microplate luminometer

Parent and IRF1 KO cells were transfected with a plasmid expressing Firefly luciferase under the control of IFNβ promoter and a plasmid constitutively expressing Renilla luciferase. Cells were then transfected with poly I:C and processed using Dual Glo luciferase assay kit (Promega) as per the manufacturer's instruction. Relative light unit was examined using a Glowmax Navigator Microplate Luminometer (Promega) at 6 h and 24 h. Firefly luciferase expression was normalized to Renilla luciferase expression.

For this assay 2–2.5 × 10⁴ HEK 293 T cells were seeded out per well of a 96-well plate (Eppendorf, Hamburg, Germany) by the liquid handling system epMotion 5075 (Eppendorf, Hamburg, Germany). HEK 293 T cells were transfected with 50 ng/well reporter vector with or without 3′UTR and 200 ng/well pSG5 empty vector or pSG5-miR-34a expression plasmid. Forty-eight hours after transfection cells were lysed and the cell lysates were prepared according to manual of the Dual-Luciferase^® Reporter Assay System (Promega, Madison, USA) and measured with the GlowMax navigator microplate luminometer (Promega, Madison, USA).

To enhance the reproducibility and replicability of results, we utilized the automatic liquid handling system epMotion 5075 (Eppendorf, Hamburg, Germany) for the Dual-Luciferase Reporter Assay in this study. Specifically, 3.6 × 10⁴ HEK-293T cells were seeded per well of the 96-well culture plates (Eppendorf) and were transfected 24 h later with 1 µL/well PolyFect transfection reagent (Qiagen, Hilden, Germany) containing 50 ng/well of each reporter vector and 200 ng/well of each expression vector. For each run, empty vector constructs (pMIR-empty and pSG5-empty) were included as a control, and all plasmid combinations were transfected in technical duplicates. After 48 h, cells were lysed and measured using the Dual-Luciferase Reporter Assay System manual with the GlowMax navigator microplate luminometer (Promega, Madison, WS, USA). We performed Luciferase assays of the WT 3’UTRs in four independent experiments and compared WT to mutated binding sites within the 3′UTR in three independent experiments. As a positive control, we re-evaluated the downregulation of four target genes (PFKFB4, HMMR, UBQLN3, and ODF2) previously studied by overexpressing microRNA-23a/b-3p using this automated technique, as shown in Figure S1.