**5.1. Gene expression profiles of dietary-induced hyperlipidemia for whole blood RNA**

RNA analyses were conducted on blood samples obtained at weeks 10, 19, and 27 of the feeding periods to characterize the dietary effects on gene expression profiles in whole blood and white blood cells of miniature pigs. Each RNA sample was analyzed by aporcine gene expression microarray consisting of 43603 oligonucleotide probes.

Variation in correlation coefficients among individuals on the same diet and between different diet groups was evaluated. Pearson correlation coefficients were used for the correlation analysis. Correlation coefficients for 23 microarrays in total were obtained for a normalized signals log-scale after excluding "absent" spots, definition of "absent" were described in Materials and Methods. A color-coded pairwise correlation matrix is displayed in Figure 7.

The correlation coefficients of whole blood expression profiles within the same diet groups were 0.97 (0.01) (mean (standard deviation; SD)), and 0.94 (0.05) for the control, HFCD whole blood at 10 weeks, 0.94 (0.03), and 0.93 (0.06) at 19 weeks, and 0.95 (0.02), and 0.95 (0.03) at 27 weeks, respectively. Using Fisher's Z-transformation to normalize the correlation distributions, no significant differences in correlation coefficients among dietary groups were observed at any period during the treatments. This indicates uniformity of dietaryinduced hyperlipidemia for our protocols.

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**Figure 7. Correlation matrix of dietary-related gene expression profiles of whole blood**. This colorcoded correlation matrix illustrates pairwise correlations between the levels of gene expression in individuals. Probe sets with normalized signals (log-transformed and scaled) were used to calculate correlations between 23 arrays using Pearson correlation coefficient; signals flagged as "absent" were

**Figure 8. Correlation matrix of dietary-related gene expression profiles of whole blood and white blood cells.** This color-coded correlation matrix illustrates pairwise correlations between the levels of gene expression in individual at feeding period at week 27. Probe sets with normalized signals (logtransformed and scaled) were used to calculate correlations between 15 arrays using Pearson correlation coefficient; signals flagged as "absent" were excluded. The color scale at the bottom indicates the

strengths of the correlations.

excluded. The color scale at the bottom indicates the strengths of the correlations.

The whole blood correlation coefficients among the different diet groups were 0.95 (0.04) for control vs. HFCD at 10 weeks, 0.93 (0.03) at 19 weeks, and 0.95 (0.03) at 27 weeks, respectively.

## **5.2. Assigning known functions to gene expression - Gene ontology annotation**

Up- and down-regulated genes were identified and classified these according to function using information from the Gene Ontology (GO) Database to understand the observed differences in whole blood gene expression profiles for the different dietary groups. Topranked genes with fold changes in expression greater than 2.0 (p < 0.05) and less than 0.5 (p < 0.05) were selected at 10, 19, and 27 weeks. As a result, the GO categories of many genes up-regulated at the end of the 19-week dietary period were related to nucleotide binding (GO: 0000166, GO: GO: 0005524, 0005525, GO: 0017076, GO: 0019001, GO: 00032553, GO: 00032555, GO: 0032561), and catabolic processes (GO: 0009057, GO: 0019941, GO: 0030163, GO: 0043632, GO: 0044257, GO: 0044265,). Many genes down-regulated after 27 weeks were in the GO categories related to biological adhesion (GO: 0007155, GO: 0022610).
