cRNA (25 mL) was fragmented using 53 fragmentation buffer (Mirus) at 948C for 15 min so that the effects of fragmentation on signal intensity could be evaluated on the microarray. Without further puriﬁcation steps that compromised signal intensity, 6 mL (;120 ng) of the labeled cRNA samples in 24 mL hybridization solution were hybridized overnight with the microarray (CombiMatrix) at 428C as previously described (29). Gene expression analysis. Distal colonic tissue samples were removed from RNAlater stabilization reagent (Sigma), placed in 1 mL of TRIzol Reagent (Invitrogen), and homogenized using beads (mix of 2.5-mm glass and 0.1- to 1.0-mm diameter silicon-zirconian beads) in a MiniBeadbeater- 8 (BioSpec Products). Total RNA was extracted (using TRIzol Reagent manufacturer’s instructions) and further puriﬁed using RNAeasy mini spin columns (QIAGEN) with a DNase on-column digestion per the manufac- turer’s instructions. RNA integrity was checked using a Bioanalyzer 2100 (Agilent Technologies) and quantiﬁed using a NanoDrop ND-1000 Spec- trophotometer (Thermo Fisher Scientiﬁc). Dietary effects on colonocyte DNA damage and repair pathways were determined by examining candidate rat genes from the 9 replication and repair pathways listed in the Kyoto Encyclopedia of Genes and Genomes (32) and other related genes (e.g., for DNA topological change and chromatin remodelling) listed in Gene Ontology (33). Whole genome expression arrays were used (Affymetrix Rat Gene 1.0 ST Array, Affymetrix). Labeling and hybridization protocols were performed using 100 ng of total RNA using the standard Affymetrix procedure without the ribosomal RNA reduction step. The hybridized probe arrays were stained with a streptavidin and phycoerythrin conjugate, scanned (GeneArray scanner), and analyzed by Affymetrix Microarray Suite software. The relative levels of the differentially expressed genes of interest (as identiﬁed by microarray data analysis) were determined by qRT-PCR on RNA that had been reverse transcribed (method described in Supplemental Text; see Supplemental Table 3 for primer details). Statistical analyses. Data on body, organ, and digesta weights, digesta pH, and biochemistry are presented as the mean 6 SEM for each treatment group. The effect of treatments was determined by 1-way ANOVA and differences between treatments were analyzed post hoc by Tukey’s honestly signiﬁcant difference test. Relationships between measures were determined by Pearson correlation analysis. Analyses were performed using a SigmaStat statistical software program (SigmaStat 2.0 for Windows, SPSS). For the gene expression microarray array data, 1-way ANOVA and post hoc pairwise comparisons using Tukey’s honestly signiﬁcant difference test were performed on the normalized gene expression data using R (R version 2.11.0, R Foundation for Statistical Computing). The gene expression qRT-PCR data were normalized (to the endogenous controls) and analyzed for differential expression by the same method. Principal components analysis (PCA) was carried out on normalized array signal intensities from the microbiota microarray using the Genespring 7.3 software program (Agilent Technologies). P , 0.05 was considered signiﬁcant for all datasets. Results There were no signiﬁcant differences in ﬁnal body weight between the treatment groups consuming the Western diet, and body weights of all but the rats consuming W-HAMS and W- HAMSB were signiﬁcantly greater than for the rats fed C-LAMS (Table 1). Group food intake (measured on a weekly basis) did not differ between any of the treatments (data not shown). Cecal tissue and digesta weights were signiﬁcantly higher and digesta pH was signiﬁcantly lower in the W-HAMS and W-HAMSB groups relative to the C-LAMS, W-LAMS, and W-LAW groups. However, whereas cecal tissue weight was signiﬁcantly higher, digesta mass and pH were unaffected in rats fed W-HAW (Table 1). Although the weight of colonic tissue was unaffected by dietary treatment, the weight of colonic digesta was signiﬁcantly higher in the W-HAMSB group than in all other groups. Colonic digesta pH was signiﬁcantly lower in the W-HAMS and W-HAW groups, but not the W-HAMSB group, relative to the C-LAMS, W-LAMS, and W-LAW groups (Table 1). Dietary treatment did not affect the weights of liver, spleen, and heart, but kidney weight was signiﬁcantly higher in the W-HAMSB and W-LAW groups relative to the C-LAMS group (Table 1). Testicular (epididymal) fat pad weight was signiﬁcantly greater in the W- LAW group than in the C-LAMS group (Table 1). The cecal and colonic SCFA pools and hepatic portal venous SCFA concentrations are presented in Table 2. The levels in the cecum, colon, and portal vein did not differ signiﬁcantly among the C-LAMS, W-LAMS, or W-LAW groups. The total and individual SCFA pools within the cecum were signiﬁcantly greater than W-LAMS when W-HAMS, W-HAMSB, or W-HAW TABLE 1 Effects of including different sources of starch in a Western diet moderate in fat and protein on ﬁnal body, gut tissue, digesta, and organ weights, and digesta pH of rats1 Variables Body weight, g Cecum weights, g Tissue Digesta Cecum pH Colon weights, g Tissue Digesta Colon pH Mucus thickness, mm Organ weights, g Liver Spleen Heart Kidney Epididymal fat C-LAMS 479 6 11a 1.22 6 0.08a 1.57 6 0.15a 6.79 6 0.10a 1.86 6 0.13 1.79 6 0.23a 6.74 6 0.06a 600 6 50 16.0 6 0.5 0.76 6 0.04 1.30 6 0.03 1.34 6 0.03a 5.00 6 0.51a W-LAMS 538 6 14b 1.01 6 0.08a 1.36 6 0.11a 6.84 6 0.09a 1.73 6 0.11 1.82 6 0.13a 6.71 6 0.18a 680 6 50 17.8 6 0.8 0.84 6 0.11 1.47 6 0.06 1.47 6 0.05ab 7.43 6 0.72ab W-HAMS 523 6 8ab 1.80 6 0.13b 3.35 6 0.48b 6.34 6 0.11b 1.98 6 0.20 2.25 6 0.22a 6.12 6 0.05b 550 6 40 17.5 6 0.6 0.73 6 0.03 1.35 6 0.03 1.46 6 0.04ab 6.42 6 0.43ab W-HAMSB 525 6 17ab 1.90 6 0.16b 3.97 6 0.35b 6.22 6 0.07b 1.87 6 0.19 3.50 6 0.41b 6.38 6 0.08ab 580 6 50 18.5 6 1.3 0.80 6 0.04 1.34 6 0.04 1.56 6 0.05b 7.27 6 0.83ab W-LAW 558 6 10b 1.13 6 0.09a 1.75 6 0.10a 6.68 6 0.07a 2.18 6 0.11 1.81 6 0.18a 6.71 6 0.05a 590 6 40 18.7 6 0.6 0.86 6 0.10 1.40 6 0.03 1.55 6 0.04b 8.03 6 0.40b W-HAW 547 6 13b 1.70 6 0.13b 2.36 6 0.27a 6.62 6 0.11a 2.07 6 0.21 1.85 6 0.24a 6.28 6 0.12b 600 6 60 17.6 6 0.8 0.75 6 0.15 1.37 6 0.02 1.51 6 0.05ab 7.42 6 0.62ab 1 Values are mean 6 SEM, n = 10. Means in a row with superscripts without a common letter differ, P , 0.05. C-LAMS, control diet containing low amylose maize starch; W-HAMS, Western diet containing high amylose maize starch; W-HAMSB, Western diet containing butyrylated high amylose maize starch; W-HAW, Western diet containing high amylose wheat ﬂour; W-LAMS, Western diet containing low amylose maize starch; W-LAW, Western diet containing low amylose wheat ﬂour. 834 Conlon et al.