Comprehensive search for Target genes of miR-199a-3p, miR-99b-3p and miR-140-5p in neural cells.

the expression of 167, 86, and 44 mRNAs was found to be significantly downregulated in Neuro-2A cells, primary astrocytes and BV-2 cells, respectively. Overall design: Equal amounts of three custom miRNA mimics (miR-199a-3p, miR-99b-3p and miR-140-5p) (Horizon) (Supplementary Table 16) and Dharmafect 1 (T-2001, Horizon) were mixed to form a complex using PS-free serum-free medium. The mimic-Dharmafect1 complex was then diluted with complete medium to 25 nM. For the control mimic, cel-miR-67 was used (Horizon). After cultivation in PS-free medium (37°C, 5% CO2, 24 h), the cells were incubated in a transfection medium for 24 h. Following transfection, RNA was extracted using QIAzol lysis reagent. Finally, the obtained RNA samples were analysed by NGS (DNA Chip Research Institute, Tokyo, Japan). Cell seeding was conducted at 6.0×105 cells/mL for Neuro-2A cells, at 9.0×105 cells/mL for primary astrocytes and at 3.0×105 cells/mL for BV-2 cells.