Franny Jongbloed

98 CHAPTER 4 numbers used for phenotypical and transcriptional endpoints is summarized in Table S5. Total RNA was extracted using QIAzol lysis Reagent and miRNAeasy Mini Kits (QIAGEN, Hilden, Germany), following Qiagen protocol. Addition of wash buffers RPE and RWT (QIAGEN) was done mechanically by using the QIAcube (QIAGEN, Hilden, Germany) via the miRNAeasy program. Isolated RNA was and stored at -80°C. The concentration of RNA was measured by Nanodrop (Thermo Fisher Scientific™, Breda, the Netherlands) and RNA quality was assessed using the 2100 Bio-Analyzer (Agilent Technologies, Amstelveen, the Netherlands) according tomanufacturer’s instructions.TheRNAqualitywas expressed as the RNA integrity number (RIN, range 0-10). RIN values of included samples ranged between 6.6 and 8.5. Hybridization to Affymetrix HT MG-430 PM Array Plates was performed at the Microarray Department of the University of Amsterdam (the Netherlands), according to Affymetrix protocols. Four to six biological replicates were used for each group. Quality control and normalization were performed using the pipeline at the www.arrayanalysis.org website (Maastricht University, the Netherlands) 26 . Normalization was done via the Robust Multichip Average (RMA) algorithm 51 . Due to a range in hybridization dates between fasting and the other diets ( i.e. September 2011 versus August 2012), normalization of the data for fasted animals and their controls was done separately. Normalization output consisted of data for 45,141 probes, with several probes corresponding to the same Gene ID. Complete raw and normalized microarray data and their MIAME compliant metadata have been deposited at the Gene expression Omnibus (GEO) database GEO GSE65656 (www. ncbi.nlm.nih.gov/geo). After normalization, outliers were found in the control SDS (control 30% DR), the 3-day fasting and 3-day fat-free diet groups, defined as a deviation in array- array intensity correlations, principal component analysis and cluster dendograms. These outliers were excluded from further analyses (Table S5). Statistical analysis Data are expressed as means ± standard error of the mean (SEM). Statistical analyses were performed using SPSS (version 21.0) and GraphPad Prism (version 5.0). Differences in serum urea concentrations were compared by Mann-Whitney U tests, food intake via the paired t-test and survival rates were analyzed by Log-rank tests. A P- value of <0.05 was considered significant. Microarray analyses were performed using the free software package R (R foundation). Gene expression data were compared using the Linear Models for Microarray Data (limma) method with correction for multiple testing using the false discovery rate (FDR) according to Benjamini and Hochberg 52 . Fold changes were expressed as the geometricmean per diet group against the corresponding ad libitum fed control group. Cutoff values for a significant difference were put at FDR <5% with fold change ≥1.5. The enrichment factor (EF) was calculated via the formula: EF=nAB/((nA×nB)/nC), where nA is the number of differentially expressed probe sets (DEPS) in experimental group A, nB the