Mirvana mimic control

HEK293T cells were seeded into 24 well plates at 1.5 × 10⁵ cells/well in DMEM + 10% FBS media. Cells were transfected 24 hours later using Lipofectamine^® 3000 Transfection Reagent (ThermoFisher Scientific), according to manufacturer`s protocol with 10 ng of either wild-type or mutant pMIR-PPP2R3A reporter plasmid containing firefly luciferase, together with 10 ng of pRL-TK plasmid (Promega, Wisconsin, USA), and 20 pmol of either miR-652 mimic (hsa-miR-652-3p; mirVana™ #MC12699, ThermoFisher Scientific) or (syn-hsa-miR-652-3p miScript mimic; MSY0003322, Qiagen), or scrambled negative control (mirVana™ control mimic #4464058, Thermofisher) were transfected per well. For blocking experiments, 40 pmol of anti-hsa-miR-652-3p miScript miRNA inhibitor (Qiagen) was added per well. Twenty-four hours after transfection, Firefly and Renilla luciferase activity were measured using Dual-Glo Luciferase Reporter Assay System (Promega). The Renilla luciferase activity was normalized to the Firefly luciferase activity. Transfections were done in triplicate, and replicated thrice. A 2-tailed T-test was performed. P-values < 0.05 were considered significant.

PCMV-MIR plasmid containing human miR-652 (MI0003667, Origene) or empty vector control (pCMVMIR, Origene) was transfected into PC3 or LNCaP cells using Lipofectamine™ 3000 transfection reagent (Life Technologies). Briefly, for 10 cm² surface area, 5 µg of plasmid DNA was transfected together with 10 µl of Lipofectamine™ 3000 and 10 µl of P3000 reagent. Cells were passaged into fresh media 48 hours post-transfection. Selection reagent, G418 (400 µg/ml), was added 48 hours post-transfection. Once stable cells were established, the GFP+ population was further selected by flow cytometric cell sorting. For transient transfection, PC3 (10⁵/ml) or LNCaP (2 × 10⁵/ml) cells were seeded 24 hours prior to transfection in 6 well plates. Immediately prior to transfection, regular media was replaced with media containing 1% FBS. Transfections were performed using Lipofectamine^® RNAiMAX transfection reagent (Thermofisher) according to manufacturer’s recommendations. For prolonged transient transfection, every 3–4 days, media was changed and cells were retransfected with 10 pmol/ml of miR-652 (hsa-miR-652-3p; mirVana™ #MC12699, Thermofisher), or negative control mimic (mirVana™ control mimic #4464058, Thermofisher). For blocking experiments, 20 pmol/ml of anti-hsa-miR-652-3p miScript miRNA inhibitor (Qiagen) were added per well.