Y lower in common marmosets than in humans while those of IL-10, IL-12b and IFN-c were significantly higher in common marmosets compared with humans. Of interest, the expression level of IL-4 was notably lower in common marmosets than humans, and was close to the detection limit. There was no statistical Terlipressin custom synthesis difference in the expression levels of the other genes tested between common marmosets and humans.Difference of CD4/CD8 ratio between humans and common marmosetsWe calculated ratios 1655472 of the expression levels of CD4 to CD8 (CD4/CD8 ratio) in human and common Tunicamycin chemical information marmoset leukocytes (Figure 5, left panel). CD4/CD8 ratios were significantly higher inhuman leukocytes compared with common marmoset leukocytes (mean 6 sd, 0.5960.22 vs. 20.4960.41, P,0.01). To confirm the difference in CD4/CD8 ratios, we examined the proportion of CD4+ and CD8+ in CD3+ T cells by flow cytometric analysis. As shown in Figure 6, the rates of CD3+ cells in the lymphocyte gate were similar between common marmosets (30 ) and humans (38 ). However, the rates of CD4+/CD3+ cells and CD8+/CD3+ cells was 36 and 61 in common marmosets, respectively, and 75 and 21 in humans, respectively. Similarly, the CD4/CD8 ratio was markedly different between common marmosets and humans (mean 6 sd, 0.5660.08 vs. 3.2260.35, P,0.01) by qPCR. This indicated a good correlation between the results from FACS analysis and that of qPCR analysis. To examine whether the CD4/CD8 ratio is affected by age, we further performed FACS analyses with PBMCs from young and old marmosets (Table 3). The result showed that the inverted CD4/CD8 ratio was fairly constant among individuals and over ages.Figure 4. The expression levels of CD antigens and cytokine genes in common marmoset and human leukocytes. The expression level of each gene is shown as a logarithmic histogram of absolute copy numbers per mg of total RNA. Means and standard deviations of eight individuals are indicated. Asterisk indicates statistically significant differences between marmosets and humans by Student’s t-test (*P value,0.05, **P value,0.01). doi:10.1371/journal.pone.0056296.gGene Expressions in Marmoset by Accurate qPCRFigure 5. The expression ratios of CD8 to CD4 (CD8:CD4) and Th1-related genes to Th2-related genes. The ratio of CD8:CD4 (left panel), IFN-c:IL-4 (middle panel) and IL-2:IL-4 (right panel) in human and common marmoset leukocytes, spleen, lymph node and thymus are shown. Significant differences in the CD8:CD4, IFN-c:IL-4 and IL-2:IL-4 ratios were found between human leukocytes and common marmoset tissues (*P,0.05). doi:10.1371/journal.pone.0056296.gDifference in T helper 1 (Th1)/T helper 2 (Th2) balance between humans and common marmosetsWe compared the ratios of expression levels of Th1-related genes (IFN-c or IL-2) and Th2-related genes (IL-4) (IFN-c:IL-4 or IL-2:IL-4 ratio) (Figure 5, middle and right panels). Both logarithmic values of the IFN-c:IL-4 and IL-2:IL-4 ratios were negative in human leukocytes whereas those of common marmoset leukocytes, spleen, lymph node and thymus indicated positive values, showing a clear difference in the Th1/Th2 balance between humans and common marmosets.DiscussionIn the present study, we evaluated the expression stability of common marmoset housekeeping genes in various tissues. To the best of our knowledge, this is the first report of a systematicevaluation of potential reference genes in common marmosets. We chose eight commonly used classical housekeeping genes. Of all gene.Y lower in common marmosets than in humans while those of IL-10, IL-12b and IFN-c were significantly higher in common marmosets compared with humans. Of interest, the expression level of IL-4 was notably lower in common marmosets than humans, and was close to the detection limit. There was no statistical difference in the expression levels of the other genes tested between common marmosets and humans.Difference of CD4/CD8 ratio between humans and common marmosetsWe calculated ratios 1655472 of the expression levels of CD4 to CD8 (CD4/CD8 ratio) in human and common marmoset leukocytes (Figure 5, left panel). CD4/CD8 ratios were significantly higher inhuman leukocytes compared with common marmoset leukocytes (mean 6 sd, 0.5960.22 vs. 20.4960.41, P,0.01). To confirm the difference in CD4/CD8 ratios, we examined the proportion of CD4+ and CD8+ in CD3+ T cells by flow cytometric analysis. As shown in Figure 6, the rates of CD3+ cells in the lymphocyte gate were similar between common marmosets (30 ) and humans (38 ). However, the rates of CD4+/CD3+ cells and CD8+/CD3+ cells was 36 and 61 in common marmosets, respectively, and 75 and 21 in humans, respectively. Similarly, the CD4/CD8 ratio was markedly different between common marmosets and humans (mean 6 sd, 0.5660.08 vs. 3.2260.35, P,0.01) by qPCR. This indicated a good correlation between the results from FACS analysis and that of qPCR analysis. To examine whether the CD4/CD8 ratio is affected by age, we further performed FACS analyses with PBMCs from young and old marmosets (Table 3). The result showed that the inverted CD4/CD8 ratio was fairly constant among individuals and over ages.Figure 4. The expression levels of CD antigens and cytokine genes in common marmoset and human leukocytes. The expression level of each gene is shown as a logarithmic histogram of absolute copy numbers per mg of total RNA. Means and standard deviations of eight individuals are indicated. Asterisk indicates statistically significant differences between marmosets and humans by Student’s t-test (*P value,0.05, **P value,0.01). doi:10.1371/journal.pone.0056296.gGene Expressions in Marmoset by Accurate qPCRFigure 5. The expression ratios of CD8 to CD4 (CD8:CD4) and Th1-related genes to Th2-related genes. The ratio of CD8:CD4 (left panel), IFN-c:IL-4 (middle panel) and IL-2:IL-4 (right panel) in human and common marmoset leukocytes, spleen, lymph node and thymus are shown. Significant differences in the CD8:CD4, IFN-c:IL-4 and IL-2:IL-4 ratios were found between human leukocytes and common marmoset tissues (*P,0.05). doi:10.1371/journal.pone.0056296.gDifference in T helper 1 (Th1)/T helper 2 (Th2) balance between humans and common marmosetsWe compared the ratios of expression levels of Th1-related genes (IFN-c or IL-2) and Th2-related genes (IL-4) (IFN-c:IL-4 or IL-2:IL-4 ratio) (Figure 5, middle and right panels). Both logarithmic values of the IFN-c:IL-4 and IL-2:IL-4 ratios were negative in human leukocytes whereas those of common marmoset leukocytes, spleen, lymph node and thymus indicated positive values, showing a clear difference in the Th1/Th2 balance between humans and common marmosets.DiscussionIn the present study, we evaluated the expression stability of common marmoset housekeeping genes in various tissues. To the best of our knowledge, this is the first report of a systematicevaluation of potential reference genes in common marmosets. We chose eight commonly used classical housekeeping genes. Of all gene.
