Molecular Characterization of Neural Cells

Genomic DNA was extracted from neurons and astrocytes by DNeasy Blood and Tissue Kit (Qiagen). Semiquantitative polymerase chain reaction (PCR)-based amplification of sex-determining region protein gene on Y chromosome (Sry) was done as previously reported [17 (link)] to identify and confirm male cells. We used the interleukin 3 (Il3) gene as an autosomal gene and an internal control for both sexes. PCR products were identified based on size (Sry, 402 base pairs (bp); Il3, 544 bp). RNA was extracted from male and female cells by Trizol extraction method (Life Technologies Inc., 15596-026) and mirVana RNA extraction kit (Thermo Fisher Scientific, AM1560), respectively. Quantitative reverse transcription PCR (qRT-PCR) for X-inactive specific transcripts (Xist) was done for female cells as previously described [17 (link),52 (link)].