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Copy number of PIK3CA gene was analyzed by quantitative real-time (qRT) PCR. For the quantitative determination of PIK3CA content relative to nDNA, primers for specific amplification of exon 20 in PIK3CA gene and nDNA-encoded ß-actin gene were selected according to previous study . Real-time PCR was then carried out on an LightCycler 480 II system (Roche Diagnostics, Germany) with a total volume of 20 µl reaction mixture containing 10 µl SYBR Green Master MIX (Takara, Japan), 8 pmol of each primers, and DNA (50 ng). The PCR conditions were 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 30 s. The threshold cycle number (Ct) values of the ß -actin gene and PIK3CA gene were determined. The copy number of PIK3CA in each tested specimen was then normalized against that of ß -actin gene to calculate the relative PIK3CA copy number. Each measurement was repeated in triplicate and 5 serially diluted control samples were included in each experiment. Copy amplification of PIK3CA gene was defined by a copy number ≥ 3. Samples with a PIK3CA/ ß -actin ratio between 1.0 and 3.0 were classified as having PIK3CA gain.. The purpose of this study is to determine the pharmacokinetics of.