Total cellular RNAs including messenger RNA (mRNA), miRNA and primary miRNA (pri-miRNA) were extracted from neurospheres using miRNeasy Mini Kit (Qiagen), and then single-stranded complementary DNA (cDNA) was synthesized using SuperScript VILO Master Mix (Life Technologies). Real-time quantitative RT-PCR (qRT-PCR) analysis of RNAs was conducted using a QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). TaqMan probes were TaqMan Assays products (Applied Biosystems). All qRT-PCR data were captured using the QuantStudio 12 K Flex software v1.2.2 (Applied Biosystems). The ratios of relative concentrations of target molecules to the GAPDH gene for mRNA and pri-miRNA, and to U6 snRNA (small nuclear RNA) for miRNA, were calculated. All the reactions were performed in triplicate, based on the standard curve method.
