The copy numbers of six CNV regions were examined by real-time quantitative PCR (Q-PCR) in 48 samples on the ABI Prism 7900HT system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Dye. The primer pairs were designed using PrimerExpress2.0 software (sequences available upon request). The endogenous control was designed to target the DEC2 gene in chromosome 12, avoiding any known structural variations including CNVs. The ΔΔCt method (User Bulletin #2 for ABI Prism 7700 Sequence Detection System; Applied Biosystems) was employed to quantify the genomic copy numbers by setting a normal number at two copies. For all CNV regions and for all samples, the quantitative copy number estimates by Q-PCR are 1.0E-6 to 3.8E-4 for zero copy genomic regions, 0.79–1.33 for one copy, 1.58–2.52 for two copies, 2.63–3.36 for three copies, 3.62–4.72 for four copies, therefore implicating the high accuracy of Q-PCR for CNV validation.
