The X Chromosome Inactivation assay was performed on genomic DNA extracted from peripheral blood as described (37). Briefly, X Chromosome Inactivation was measured by the analysis of the (CAG)n repeat in the androgen receptor gene at Xq11-q12 before and after digestion with methylation sensitive restriction enzymes HhaI and HpaII. Quantitative PCR amplification of androgen receptor gene repeat alleles was compared, with and without restriction digestion, to determine the ratio of X-active/inactive alleles.
