As MECP2/Mecp2 is an X-linked gene, it is possible that the observed changes in Mecp2 expression are due to a shift in the number of cells derived from male and female embryos. To exclude such a possibility, we determined the contribution from the male/female embryonic cells during NSC differentiation using a semiquantitative PCR-based method. Genomic DNA was extracted from each differentiation stage and subjected to PCR analysis for the presence of the Sry gene found in the Y chromosome. The autosomal gene Il3 was used an internal control. The adult male brain cortex was used as a positive control for the presence of the male genomic DNA (Additional file 4: Figure S4A). We did not observe statistically significant changes in the ratio of Sry/Il3 in the cells collected at different stages of differentiation, indicating that the ratio of male/female differentiating NSC were unchanged (Additional file 4: Figure S4B). To further confirm the contribution from the male/female gender, we tested the transcript levels of Xist gene (the gene is involved in X-chromosome inactivation) by qRT-PCR. We did not detect any significant
