To study the set of miRNAs regulated by SOX2, we subjected samples to a miRNA array analysis. Total RNA from scrambled and SOX2-knock down GSC-11 cells was reverse transcribed into cDNA using Taqman miRNA Reverse Transcription Kit (Applied Biosystems) and loaded onto a Human miRNA array panel containing 384 wells according to manufacturer’s protocol (Applied Biosystems). Quantitative miRNAs expression data were normalized with U6b housekeeping gene and quantified using ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). The microarray data from this study have been submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE115086.
For analysis of miRNA targets induced by SOX2, total RNA including small RNA was extracted from GBM1A and GBM1B expressing exogenous SOX2 (GBM1A-SOX2) using miRNeasy kit (Qiagen). The cDNA was synthesized using miScript II RT kit (Qiagen) and used to perform microarray analysis using a brain cancer miRNA PCR Array (SABiosciences, Frederick, MD, USA) according to manufacturer’s instructions. Array data were analyzed using miScript miRNA PCR array data analysis tools (Qiagen, SABiosciences).
For analysis of miRNA targets induced by SOX2, total RNA including small RNA was extracted from GBM1A and GBM1B expressing exogenous SOX2 (GBM1A-SOX2) using miRNeasy kit (Qiagen). The cDNA was synthesized using miScript II RT kit (Qiagen) and used to perform microarray analysis using a brain cancer miRNA PCR Array (SABiosciences, Frederick, MD, USA) according to manufacturer’s instructions. Array data were analyzed using miScript miRNA PCR array data analysis tools (Qiagen, SABiosciences).