Q-PCR for WC1 gene number. (A) The standard curves for primer sets of WC1s (WC1-com) and other control genes. The standard curves were constructed with a series of 10-fold dilution of cDNA derived from ex vivo PBMC. Each standard dilution was amplified by real-time QPCR in duplicate. For each primer set, CT values determined from real-time QPCR were plotted against the logarithm of their known initial gene numbers. A standard curve was generated by linear regression through these points. (B) The slopes of the standard curves for primers of WC1s (WC1-com) and other control genes. From the slopes, amplification efficiencies were also determined. (C) Primer specificities of WC1s (WC1-com) and other control genes’ primer sets. Confirmation of PCR amplification specificities by melting curve for primer sets of WC1 common (WC1-com), bovine TRDJ1, bovine GAPD, bovine IFNA, bovine IFNB, bovine IFNE, and bovine IFNW. Melting peaks were examined for WC1 common (WC1-com, solid line with empty diamond marker), bovine TRDJ1 (solid line with solid circle marker), bovine GAPD (solid line), bovine IFNA (solid line with solid square marker), bovine IFNB (solid line with solid triangle marker), bovine IFNE (solid line with empty circle marker), and bovine IFNW (solid line with empty square marker). (D) Gene numbers of bovine WC1 genes and other control genes. The breeds of tested cattle are indicated in the figure. The ΔΔCT method was applied for relative quantification. Some breeds contained more than one animal, and each evaluation was performed at least twice, yielding similar results.
Chen et al. BMC Genetics 2012 13:86 doi:10.1186/1471-2156-13-86