Dual luciferase kit reagents

Mouse Neuro2a (N2a) cells (CCL-131, American Type Culture Collection [ATCC], Manassas, VA) were seeded at 80,000 cells/well in 24-well tissue culture dishes, and the next day, cells were co-transfected with 100 ng miR-181 target reporter plasmid plus 4 nM either control or miR-181 pre-miR synthetic oligos (Ambion, Austin, TX), along with 100 ng either TuD-Ctrl plasmid or TuD-181 expression plasmid, using Lipofectamine 2000 (Thermo Fisher Scientific). Each combination was assayed in triplicate culture wells. At 48 h post-transfection Firefly (FF) and Renilla (R) luciferase activities were measured using the GloMax Microplate Reader and Dual Luciferase Kit reagents (Promega). Briefly, culture media was removed and 200 μL passive lysis buffer was added to each well. The plate was incubated on a shaker for 15 min at room temperature, and then 10-μL lysate was transferred to duplicate wells of a 96-well white plate. Luminescence from FF and R was determined from 5-s reads after the injection of respective substrates to each well. The R:FF ratio was calculated and adjusted relative to the control (set to “1”), and results are expressed as the mean ± SEM (n = 4/group).

Mouse Neuro2a (N2a) cells (CCL-131; American Type Culture Collection, Manassas, VA) were plated at 80,000 cells/well in 24-well tissue culture dishes and, 24 h later, were cotransfected with 100 ng miR-29 target reporter plasmid plus 10 nM either control miR-Neg or miR-29a pre-miRNA synthetic oligos (Ambion, Austin, TX), along with 100 ng either TuD-Ctrl plasmid or TuD-29 expression plasmids, using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), completed in triplicate for each treatment combination. At 48 h posttransfection, FF and R luciferase activities were measured using a GloMax Microplate Reader and Dual Luciferase Kit reagents (Promega). Briefly, culture media was removed and 200 μL Passive Lysis Buffer was pipetted into each well, and the plate placed on a shaker for 15 min at room temperature. Then, 10-μL lysate was transferred to duplicate wells of a 96-well white plate. Luminescence from FF and R was determined from 5-s reads after the injection of respective substrates to each well. The R/FF ratio was calculated and adjusted relative to the control (set to “1”), and results are expressed as the mean ± SEM (n = 4/group).