Schematic diagram of X-chromosome clonality determination used here and in HUMARA assay. X-chromosome inactivation occurs early during embryogenesis. Hence, women are a mosaic of paternal or maternal active X chromosome (Step 1). Inactive X chromosome is represented by filled red circles. For the transcriptional clonality assay, a specific exonic polymorphism is selected and genotyped (Step 2a). Allele-specific expression is determined by real-time PCR using reverse-transcribed mRNA as described in “Novel transcriptional clonality assay” (Steps 3a and 4a). Resulting amplification curve is used to estimate the ΔCt and corresponding frequencies of each allele (Step 5a). In contrast, analysis at the HUMARA locus, shown methylated in the promoter region by filled red circles (Step 2b), is initiated by restriction digestion (scissors) of genomic DNA using a methylation-sensitive endonuclease (Step 3b). After restriction digestion, PCR amplification with primers flanking both the 5′ restriction digestion site and the 3′ end of the CAG tandem repeat sequence is performed (Step 4b). Hence, only intact, methylated, inactive X-chromosome DNA is amplified. Allele-specific PCR products can be distinguished from each other based on the number of tandem CAG repeats using agarose gel electrophoresis (Step 5b).