Biotinylated miRNA Transfection and Analysis

Biotinylated miR-H25 and miR-BART7 were obtained from Eurofins MWG Operon as a miRNA duplex in which the sense filament, at the 3′ end, was labeled with biotin. A detailed map of all the synthetic miR is provided in S6 Figure. A2780 and SKOV3 cells were used with miR-H25, while A2780 and Hey cells were utilized with miR-BART7. Cells were seeded in 6 well dishes, 2×10⁶ cell/well, for 48 h without reaching full confluency. HiPerFect transfection reagent (Qiagen, Valencia, CA) was used to transfect the cells at final concentration of 5–10 nM. For each cell line, a transfection with only HiPerFect reagent was performed as negative control. Analysis was carried out using the 48.48 dynamic array (Fluidigm Corporation, CA, USA). Cytotoxicity assays were performed with the use of the ATPlite kit as previously described [35] (link). Q-PCR analysis was performed as previously described [36] (link). Western blot for ADH1B expression was performed as previously described [37] (link) using a rabbit polyclonal antibody (Thermo Fisher Scientific Pierce, Rockford, IL). A mouse anti-GAPDH (clone G-9, Santacruz Biotechnology, Dallas, TX) antibody was used as loading control.