CAA CCG TGA AAA GAT GA-3’ and 5’-CAC AGC CTG GAT GGC TAC GT-3’ (GenBank #NM031144) generate a 79 amplicon corresponding to the nucleotide 420-498 of the sequence that crosses exon 2 and exon 3. The SYBR Green PCR core reagents kit (Applied Biosystems, Foster City, CA) was used, with β-actin as endogenous control. PCR was performed in MicroAmp Optic 96-well Reaction Plates (Applied Biosystems, Foster City, CA) on an ABI PRISM 7900 Sequence Detection system (Applied Biosystems, Foster City, CA), with the condition of 2 min at 50°C, 10 min at 95°C, then 40 cycles of 15 s at 95°C and 1 min at 60°C. Each study consisted of 4 independent runs of PCR in triplicate, and each run included a standard curve, non-template control, and negative RT control. The concentrations of primers were 100 to 200 nM, and all reagents, unless indicated, were from Invitrogen (Carlsbad, CA). The levels of target gene expression were quantified relative to the level of β-actin, using standard curve method. While GAPDH and cyclophilin were also tested in some initial experiments and found to yield stable results with no response to a high-fat diet, β-actin was generally the least variable of these 3
