Mithras lb940 multimode reader

3′-UTR (untranslated region) of each of the potential miRNA target genes was amplified from genomic DNA of normal human peripheral blood lymphocytes using Phusion Taq polymerase (Thermo scientific, Pittsburgh, PA, USA). The 3′-UTRs were then cloned downstream of firefly luciferase cDNA from PGL3 vector (Promega, Madison, WI, USA) in a pcDNA 4.0 plasmid vector (Invitrogen, Life Sciences, Carlsbad, CA, USA). The genomic region encoding miR-148a was cloned into pcDNA 3.0 plasmid vector wherein the miRNA was expressed under CMV promoter in mammalian cells. HEK 293T cells were transfected with the luciferase reporter plasmid, miRNA expressing plasmid and a plasmid vector expressing EGFP fluorescent protein by Calcium phosphate BES buffer method. Luciferase activity was assessed from the total protein extracted from the transfected HEK293T cells and was normalized against the EGFP fluorescence measured using Mithras LB940 multimode reader (Berthold Technologies, Bad Wildbad, Germany).

Transient transfection was performed using TurboFect transfection reagent (Thermo scientific, Dreieich, Germany) according to manufacturer’s instructions. The ratio of DNA to TurboFect employed was 2 μg DNA per 4 μl transfection reagent. For transfection, cells were first seeded in 96-well plates at a density of 10,000 cells per well in 200 μl of medium. After 24 hours, old medium was removed and fresh medium containing the DNA/Polymer complexes was added (100 μl with an equivalent of 25 ng DNA). Three hours later, medium was exchanged and the cells received fresh medium with the supplements to be tested. Gaussia luciferase activity was determined from the supernatant 24 hours after the start of the transfection as described before [26 (link)]. Briefly, cell supernatant (5 μl) was transferred into a well of a white 96-well plate (Greiner bio-one, Frickenhausen, Germany). After an incubation of 20 minutes at room temperature, Gaussia luciferase activity was determined using a Mithras LB 940 Multimode reader (Berthold Technologies, Bad Wildbad, Germany) by injecting 50 μl of luciferase assay reagent (20 mM MOPS (3-(N-morpholino)propanesulfonic acid); 0.75 M KBr; 5 mM MgCl₂; 5 mM CaCl₂; 1 mM EDTA (ethylenediaminetetraacetic acid); 10 μM Coelenterazine; pH 7.8) to the cell supernatant, followed by a 1.6 second delay until luminescence was determined within a 0.5 s integral.