Hepatic lipogenesis gene expression analysis

Total liver RNA from mice injected with adenovirus (10⁹ pfu per mouse) encoding apoC-III variants or empty vector was isolated with TriPure reagent (Roche), as per the manufacturer’s instructions. After removal of genomic DNA, the first-strand cDNA synthesis was performed by using a QuantiNova reverse transcription kit (Qiagen), and the template cDNA was diluted 1:20 with nuclease-free water. Relative mRNA expression was determined by quantitative RT-PCR using inventoried TaqMan assays (Srebf1; Mm00550338_m1, Srebf2; Mm01306292_m1, Fas; Mm00662319_m1, Scd1; Mm00772290_m1, Acc1; Mm00728460_s1, Acc2; Mm00624282_m1, Cd36; Mm00432403_m1, Hmgcr; Mm01282499_m1, Ldlr; Mm01177349_m1, Tbp; Mm00446973_m1, Pparg; Mm00440940_m1, Nr1h3 [liver X receptor a (LXRa)]; Mm00443451_m1, and βactin; Mm00607939_s1) combined with QuantiNova Probe PCR mix (Qiagen) on a Rotor-Gene-Q instrument (Qiagen). Relative expression was determined by using the ^ΔΔCt method (17), normalized to the average of both Tbp and βactin, and data were presented relative to vector control samples.