Each gene studied was amplified in a separate reaction, and each reaction was performed in duplicate. Each 20 μL reaction contained 1.0 μL of cDNA sample, 0.4μL of a mixture of a forward primer and reverse primer, 0.4μL of the analyzed genes [GAPDH (housekeeper), GHR, IGF1, IGF1R, IGFBP3, IGFBP5, BAX, and BCL2], 8.2 μL of RNA-free water, and 10.0 μL of SYBR Green Mix (Invitrogen, Burlington, ON, Canada). The expression of the target genes was normalized to that of GAPDH as calculated by the 2−ΔΔCT method (Schmittgen and Livak, 2008). The samples were then subjected to RT-PCR and subsequent analysis of the amplified gene fragments.