Nd 95uC for 5 minutes (termination of cDNA synthesis). Immediately after, the samples were cooled down and stored at 220uC.Plasma MeasurementsCholesterol level was measured in duplicate using the kit Cholesterol Chod-Pap (Roche Diagnostics GmbH, Germany) in accordance with the protocol of the manufacturer. Calibrator (C.f.a.s from Roche Diagnostics GmbH) and controls (Wako Control Serum I and II from Wako Chemicals GmbH, Germany) were included in the analysis. The intra- and interassay coefficients of variation were 3.6 and 6.0 , respectively.Statistical AnalysisAll statistical analyses were performed using SPSS 20.0 (IBM Corp., USA). Graphs have been constructed using GraphPad Prism version 5.0c for Mac OS X (GraphPad Software Inc., USA). All results were log-transformed to obtain Gaussian distribution as confirmed by one-sample ��-Sitosterol ��-D-glucoside Kolmogorov-Smirnov test. Comparisons between the different groups were performed by Student’s t-test for independent samples and Bonferroni correction of p-values was applied by multiplying the acquired p-values. Univariate linearIdentifying the Optimal Reference GenesThe optimal reference genes for the study were selected from a panel of twelve common endogenous control genes (prefabricated panel of primer-mixes from TATAA Biocenter, Sweden). All candidate genes were tested by quantitative realFDG and Gene Expression in Murine AtherosclerosisFigure 1. CT, fused PET/CT, and PET images. A Contrast-enhanced CT image. B Fused PET/CT image. C PET image. All images are in sagittal view. doi:10.1371/journal.pone.0050908.gregression was performed between the molecular markers (gene expression) and SUVmean-values and p-values were Bonferroni corrected. Markers with significant correlation (R) were subsequently included in a multivariate linear regression model with stepwise backward elimination of the least significant marker. Data are reported as mean6SEM (standard error of mean) unless otherwise indicated and p,0.05 was considered statistically significant.Results Uptake ofF-FDG in the Vessel WallThe uptake of order NT 157 18F-FDG measured using PET and gamma counting, respectively, is shown in Figures 3a and 3b. The uptake of 18F-FDG measured using PET (Figure 3a) was not significantly different in the groups receiving normal chow for 24 and 32 weeks compared to the 0 weeks group. However, the 8 and 16 weeks groups showed a small decrease in the uptake compared to the 0 weeks group with a fold change of 0.84 (p = 0.013) and 0.78 (p = 0.0012), respectively. The high-fat Western diet had a marked effect upon the uptake of 18F-FDG measured by PET and from 16 weeks, SUVmean was significantly higher compared to 0 weeks group. The fold change was 1.73 after 16 weeks (p = 0.0011), 1.82 after 24 weeks (p,0.001) and 2.23 after 32 weeks (p,0.001) compared to 0 weeks.When comparing mice on high-fat Western diet with mice on normal chow of the same age, a significant higher 18F-FDG uptake measured by PET was seen after 16 weeks of dieting compared to non-dieting (2.21 fold; p,0.001). The 18F-FDG uptake was 2.02 fold higher at 24 weeks on diet compared to non-diet (p,0.001). At 32 weeks, a 2.29 fold higher level was seen (p,0.001). The 18F-FDG uptake measured by gamma counting showed the same pattern (Figure 3b) as measured by PET, except, the uptake did not differ significantly in the chow-fed groups compared to the 0 weeks group. The high-fat Western diet had a marked effect upon the 18F-FDG uptake measured by gamma counting from 16 weeks.Nd 95uC for 5 minutes (termination of cDNA synthesis). Immediately after, the samples were cooled down and stored at 220uC.Plasma MeasurementsCholesterol level was measured in duplicate using the kit Cholesterol Chod-Pap (Roche Diagnostics GmbH, Germany) in accordance with the protocol of the manufacturer. Calibrator (C.f.a.s from Roche Diagnostics GmbH) and controls (Wako Control Serum I and II from Wako Chemicals GmbH, Germany) were included in the analysis. The intra- and interassay coefficients of variation were 3.6 and 6.0 , respectively.Statistical AnalysisAll statistical analyses were performed using SPSS 20.0 (IBM Corp., USA). Graphs have been constructed using GraphPad Prism version 5.0c for Mac OS X (GraphPad Software Inc., USA). All results were log-transformed to obtain Gaussian distribution as confirmed by one-sample Kolmogorov-Smirnov test. Comparisons between the different groups were performed by Student’s t-test for independent samples and Bonferroni correction of p-values was applied by multiplying the acquired p-values. Univariate linearIdentifying the Optimal Reference GenesThe optimal reference genes for the study were selected from a panel of twelve common endogenous control genes (prefabricated panel of primer-mixes from TATAA Biocenter, Sweden). All candidate genes were tested by quantitative realFDG and Gene Expression in Murine AtherosclerosisFigure 1. CT, fused PET/CT, and PET images. A Contrast-enhanced CT image. B Fused PET/CT image. C PET image. All images are in sagittal view. doi:10.1371/journal.pone.0050908.gregression was performed between the molecular markers (gene expression) and SUVmean-values and p-values were Bonferroni corrected. Markers with significant correlation (R) were subsequently included in a multivariate linear regression model with stepwise backward elimination of the least significant marker. Data are reported as mean6SEM (standard error of mean) unless otherwise indicated and p,0.05 was considered statistically significant.Results Uptake ofF-FDG in the Vessel WallThe uptake of 18F-FDG measured using PET and gamma counting, respectively, is shown in Figures 3a and 3b. The uptake of 18F-FDG measured using PET (Figure 3a) was not significantly different in the groups receiving normal chow for 24 and 32 weeks compared to the 0 weeks group. However, the 8 and 16 weeks groups showed a small decrease in the uptake compared to the 0 weeks group with a fold change of 0.84 (p = 0.013) and 0.78 (p = 0.0012), respectively. The high-fat Western diet had a marked effect upon the uptake of 18F-FDG measured by PET and from 16 weeks, SUVmean was significantly higher compared to 0 weeks group. The fold change was 1.73 after 16 weeks (p = 0.0011), 1.82 after 24 weeks (p,0.001) and 2.23 after 32 weeks (p,0.001) compared to 0 weeks.When comparing mice on high-fat Western diet with mice on normal chow of the same age, a significant higher 18F-FDG uptake measured by PET was seen after 16 weeks of dieting compared to non-dieting (2.21 fold; p,0.001). The 18F-FDG uptake was 2.02 fold higher at 24 weeks on diet compared to non-diet (p,0.001). At 32 weeks, a 2.29 fold higher level was seen (p,0.001). The 18F-FDG uptake measured by gamma counting showed the same pattern (Figure 3b) as measured by PET, except, the uptake did not differ significantly in the chow-fed groups compared to the 0 weeks group. The high-fat Western diet had a marked effect upon the 18F-FDG uptake measured by gamma counting from 16 weeks.