We performed TaqMan gene copy number assays using quantitative PCR following a previously described method [24]. All primers and TaqMan probes were designed with Primer Express software v2.0 (Applied Biosystems, Tokyo, Japan). The commercially available RNase P assay (Applied Biosystems, Tokyo, Japan) was used as a copy number reference gene. All assays were performed on an ABI 7900HT (Applied Biosystems, Tokyo, Japan) using TaqMan Gene Expression PCR Master Mix (Applied Biosystems, Tokyo, Japan) with 5 ng of genomic DNA in a 10 l reaction. The cycling conditions were the following: 95 degrees Celsius for 10 min for the initial denaturation and enzyme activation, followed by 40 cycles of 95 degrees Celsius for 15 sec and 60 degrees Celsius for 1 min. The copy number was calculated using the comparative Ct method [25], in which Ct indicates the threshold cycle.
