Real-Time PCR was carried out using SybrGreen chemistry and the ABI Prism 7300 Sequence Detection System (Applied Biosystems Inc. Foster City, CA). The amplification primers were designed using Vector NTI (Invitrogen, Carlsbad, CA). Total RNA, isolated for the microarray analyses, was treated with DNase I for these analyses. Following reverse transcription of the RNA (SuperScript™ III First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA), an aliquot of each reverse transcription reaction was amplified in triplicate. This reaction was repeated to generate 6 values for each test group. Two control reactions were run for each RNA preparation: 1) a reverse transcription and PCR reaction with no added RNA to control for contamination of the reagents; and 2) a PCR reaction without the reverse transcription reaction in the presence of RNA to detect DNA contamination of the RNA preparation. To correct for sample-to-sample variation, an endogenous control (GAPDH) was amplified with the target and served as an internal reference to normalize the data. Relative quantification of data from the ABI Prism 7300 Sequence Detection System was performed using the standard curve method
