Yamamoto et al. BMC Genomics 2012, 13:335 http://www.biomedcentral.com/1471-2164/13/335 Page 4 of 15 Figure 2 Venn diagram showing differential expression of genes in the LI, SI, LIV, and SPL of 0WexGF vs. GF, 5WexGF vs. GF, and SPF vs. GF mice. Upper row: The numbers of probe sets differentially expressed (p < 0.05). A total of 1498 probes sets were differentially expressed in the LI, 2711 in the SI, 274 in the LIV, and 1805 in the SPL. The greatest number of differentially-expressed probe sets (2188) was observed for SPF mouse SI. Only 22 ~ 36 probe sets were specifically co-regulated in all 3 comparisons in each tissue. Bottom row: The numbers of the GO BP categories having integrated p values of less than 0.05 are shown. In the LI, of the 38 overrepresented categories, 31 were specific for 0WexGF mice. In the SI, 202 of the 204 categories were specific for SPF mice. In the SPL, 160 of the 170 categories were specific for 5WexGF mice. No GO BP categories were overrepresented in the LIV. the gene set enrichment analysis program MGP to the signal pathway database of TRANTHPATH. Figure 7, Figure 8, and Figure 9 show the signal pathways overre- presented in LI, SI and SPL. TLR, Rac1, and IFN-α sig- naling were commonly overrepresented in the LI of SPF and 0WexGF mice (note that “dsRNA/TLR3” pathways are closely interconnected with the pathways for IFN-α production) and epidermal growth factor (EGF) and nerve growth factor (NGF) signaling were common in the LI of 0WexGF and 5WexGF mice. There was low commonality between 5WexGF and SPF. In 5WexGF mice, specific alteration of gene expression for chemo- taxis was indicated. The lists for SI differed from those of LI in SPF and 0WexGF mice (Figure 8). However, sur- prisingly, the list for SI seemed essentially the same as that of LI in 5WexGF mice (Figure 7 and Figure 8). In all 3 groups, the profiles of SI were characterized by large scale alteration of EGF, platelet-derived growth fac- tor (PDGF) and NGF/neurotrophin signaling. Overre- presentation of TLR pathway and Rac1 signaling was observed in 0WexGF SI but not in SPF SI. In SPL, only a few overrepresented signal pathways passed the statis- tical criteria (q < 0.001), but overrepresentation of TLR pathway and Rac1 signaling in 0WexGF mice and apop- tosis pathway signaling in 5WexGF mice was noted. No pathways reached statistical significance of q < 0.001 in LIV. Investigation of TLR signaling by quantitative RT-PCR Gene set enrichment analysis provides useful informa- tion about the statistical significance of changes in the expression of gene sets. However, the analysis does not provide information on the degree and direction of change of expression for any particular gene. Further- more, it is unlikely that any existing database would in- clude all of the signaling pathways involved in complex physiological and pathophysiological events. For exam- ples, "TLR pathway" in the TRANTPATH database seemed to contain relatively little information on TLR7 and TLR9. Therefore, in order to obtain more informa- tion regarding differences in TLR signaling, we investi- gated the expression of various TLR-related genes by real time RT-PCR analysis in LI of the 4 groups of mice. The results are summarized in Figure 10 in terms of the difference of gene expression compared with SPF mice. The status of enteric microbiota (absence, presence, and