Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat: Liver Transcriptome Changes in Study 2

Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 5 and 10 h where the rats were dosed with 2 ml/kg of corn oil vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 150-bp paired-end sequencing according to the manufacturer’s protocol (Illumina NovaSeq6000, San Diego, CA). Results: Conclusions: There were 100 genes that could be mapped to the rat metabolic network iRno, with significant changes in expression between 5 and 10 h of fasting in the top 10% by effect size parameter b (b > 0.6). Liver mRNA profiles of 5- and 10-h fasted Sprague-Dawley rats were generated by RNA-seq.