Quantitative Real-Time PCR for Liver Gene Expression

Total RNA was extracted from frozen liver that had been homogenized in TRI Reagent (Ambion Diagnostics, Austin, TX, USA), using the RNeasy Mini Kit (Qiagen N.V., Hilden, Germany). RNA concentration and purity were confirmed using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and reverse transcription PCR was carried out using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA). Quantitative polymerase chain reaction (qPCR) was carried out using SYBR Green dye and the StepOne Plus PCR machine (Applied Biosystems). Primers were designed using Primer3 [22 (link)] to the rat genome; primer sequences and GenBank references are included in Supplementary Table S1. Primers were designed to be intron-spanning to avoid amplification of genomic DNA. Product sizes and primer specificity were confirmed using classical PCR and gel electrophoresis before qPCR and melt-curves following qPCR. All qPCR results were adjusted to two reference genes (RPLP0 and GAPDH) using GeNorm for Microsoft Excel [23 (link)] and are presented as fold change in arbitrary units relative to the Gen0-C group, according to the 2^−ΔΔCT method [24 (link)].