Mirvana mimic mirna control 1

HEK293T cells were seeded at 1 × 10⁴ cells/well in opaque 96-well plates and incubated for 24 h at 37°C in a CO₂ incubator. mirVana miRNA mimics were co-transfected in triplicate with psiCHECK-2 (Promega, C8021) vectors using Lipofectamine 2000 (Invitrogen, Thermo Fisher, 11668-019) according to manufacturer’s protocol at the indicated final concentration for the mimic and 50 ng of plasmid DNA for the vector containing the miR-T cassette, in biological and technical triplicate. Each experiment also included a negative mirVana mimic miRNA control #1 (Invitrogen, Thermo Fisher, 4464058), a random sequence of miRNA mimic molecule that serves as a baseline for evaluation of the transcriptional regulation control and experimental miRNA mimic on target gene expression.³⁸ (link) A control negative (Neg) mimic with little or no off-target effect was provided by the manufacturer.

To control for transfection efficiency, we used firefly luciferase (fluc) expression to normalize for Renilla luciferase (rluc) expression. The dual-luciferase assay was performed with the SpectraMax DuoLuc Reporter Assay Kit (Molecular Devices, San Jose, CA, USA, R8361). All reagents were prepared as described by the manufacturer. Twenty microliters of 1X passive lysis buffer was added to each well for 15 min at 250 rpm on a plate shaker. Using the SpectaMax i3x Dual-Luc injector cartridge, automated injection of 100 μL of the fluc reagent was added to each test sample, with a 2 s equilibration time and measurement of luminescence with a 5 s integration time, followed by addition of 100 μL of the rluc reagent and firefly quenching, 2 s equilibration time, and measurement of luminescence with a 5 s integration time. The data are represented as the ratio of firefly to Renilla luciferase activity (fluc/rluc). Fluc expression is controlled by the miR-T cassette, and rluc serves as the control for transfection efficiency. The data were normalized to the negative control (miR-T vector and negative mirVana mimic miRNA control #1, Invitrogen, Thermo Fisher, 4464058).

A253 cells were seeded at 4 × 10⁴ cells/well in 48-well plates and incubated for 24 h in a CO₂ incubator. mirVanamiRNA mimics were transfected using RNAiMax (Invitrogen, Thermo Fisher) according to the manufacturer’s protocol at a final concentration of 6.7 nM in biological and technical duplicate. Each experiment also included a negative mirVana mimic miRNA control #1 (Invitrogen, Thermo Fisher, 4464058), a random sequence miRNA mimic molecule that serves as a baseline for evaluation of the control and experimental miRNA mimic on target gene expression.³⁸ (link) This mimic with a random sequence has been tested in human cell lines and does not produce any off-target effects. All mimic sequences are listed in Table S6.