X chromosome inactivation (XCI) assay
The HUMARA (Human Androgen Receptor) assay (Allen, Zoghbi, Moseley,
Rosenblatt, & Belmont, 1992; Bertelsen, Tumer, & Ravn, 2011;
Echevarria et al., 2016) was used to determine the ratio of inactivated
paternal vs maternal X chromosomes. Briefly, gDNA samples (0.25 ug) were
incubated with 10 U HpaII (New England Biolabs) at 37°C overnight
followed by heat inactivation of the enzyme at 80°C for 20 min. The
highly polymorphic androgen receptor promotor region of the HpaII
treated samples and untreated control samples were amplified with 0.2 μM
forward primer 5’-FAM -TCCAGAATCTGTTCCAGAGCGTGC-3’ and 0.2 μM
reverse primer 5’-TCCAGAATCTGTTCCAGAGCGTGC-3’ with 0.2 mM dNTP, 1.5 mM
MgCl2 and 0.025 U AmpliTaq Gold DNA Polymerase (Thermo
Fisher) by PCR amplification (initial denaturation at 95°C for 6 min
followed by 35 cycles of 40 s at 95°C, 30 s at 60°C, and 1 min at 72°C
and a final extension step at 72°C for 2 min). After adding the GeneScan
500 LIZ dye Size Standard (Thermo Fisher), the mixtures were analyzed on
an ABI 3730 DNA Analyzer with GeneMapper software 3.7 (Applied
Biosystems). XCI ratio was estimated by the area of the smaller base
size (XC1) and larger base size (XC2)
peaks, with formula as follows: peak area of [(XC1digested/XC1nondigested)
/ ((XC1digested/XC1nondigested) + (XC2digested/XC2nondigested )) ].