Two microliters of 5-fold dilution of RT product was used for PCR in a final volume of 25 μL containing 12.5 μL SYBR Green Real-time PCR Master Mix (TOYOBO Ltd., Japan) and 0.2–0.8 μMof each forward and reverse primers for D1, D3, GHR and IGF-1 (Table 3). Chicken β-actin mRNA was used as a reference gene for normalization purposes.