Quantitative Real-Time RT-PCR was performed on a select subset of relevant genes chosen from genes showing significant differential expression by gene array analysis. A preliminary experiment was performed to select an appropriate endogenous control gene for RT-PCR analysis. Thirty-two isolated RNA samples (2 μg) from the C4, A1, A4, and AI4 treatment groups (n = 8/group) were first DNase-treated (Turbo DNA-free kit Ambion Inc., Austin, TX) and reverse transcribed into cDNA (High Capacity cDNA RT kit Applied Biosystems, Foster City, CA). Residual RNA was removed by RNase H treatment and samples were cleared of residual RNA fragments and dNTPs (MEGAclear-96 kit, Ambion Inc.). UV spectroscopy was performed to quantify cDNA samples (NanoDrop Technologies, Inc., Wilmington, DE). Thirty ng of each cDNA sample was run on 4 TaqMan Low Density Array Endogenous Control Panels on the Applied Biosystems 7900HT Gene Expression Array System, used to measure relative expression of 16 common endogenous control genes. Data analysis was performed using ABI’s Sequence Detection Systems (SDS) software 2.1, and the gene showing the least deviation in Ct values across all samples (β-Tubulin, B2M)
