Yamamoto et al. BMC Genomics 2012, 13:335 http://www.biomedcentral.com/1471-2164/13/335 Page 12 of 15 (See figure on previous page.) Figure 10 Summary of the results of RT-PCR of TLR-related genes in LI. The molecules involved in TLR signaling are shown in two clusters: one leads to the induction of proinflammatory cytokines in an NFκB-dependent manner, and the other leads to the production of type 1 IFN in an NFκB-independent manner. Expression levels are represented by columns indicating the relative value of each group normalized to the maximum value in the four groups. Number represents fold-change to the expression level in SPF. Brown, GF; ocher, 0WexGF; light green, 5WexGF; dark green, SPF. Differences compared with SPF mice were analyzed by Dunnet test. * p < 0.05. RNA extraction from mouse tissue Mice were sacrificed and the LI, SI, LIV, and SPL were harvested for preparation of total RNA. Each frozen sam- ple was homogenized in 1 ml/0.1 g tissue of TRI Reagent (Sigma-Aldrich Japan, Tokyo, Japan) with a Polytron tis- sue homogenizer (Kinematica, Littau-Lucerne, Switzer- land) and incubated for 10 minutes at RT. Chloroform (0.2 ml/1 ml TRI Reagent) was added to the samples and the suspensions were centrifuged at 13,200 × g for 15 min at 4 °C. The water phase was transferred to a new tube and the RNA was prepared using a conventional isopro- panol/ethanol precipitate technique. Microarray analysis Total RNA extracted from mice (n = 3 per each group) were re-purified using RNeasy spin columns (Qiagen, Valencia, CA) according to the manufacturer's instruc- tions. All samples were monitored using an Agilent Table 2 Top 25 GO categories overrepresented in SI of SPF mice compared to GF mice GOID Term Number of Probes GO:0015992 proton transport 67 p_int < 1.0E-18 < 1.0E-18 < 1.0E-18 < 1.0E-18 < 1.0E-18 1.11E-16 2.78E-15 5.32E-13 1.05E-12 2.27E-11 2.41E-11 3.77E-11 3.84E-11 5.23E-11 5.23E-11 5.23E-11 5.23E-11 6.78E-11 1.00E-10 4.44E-10 7.76E-10 1.06E-09 1.15E-09 2.04E-09 2.27E-09 8.14E-05 GO:0015986 GO:0006099 GO:0016192 GO:0006631 GO:0045454 GO:0006754 GO:0006096 GO:0016481 GO:0042254 GO:0009987 GO:0001501 GO:0006461 GO:0021799 GO:0021813 GO:0021589 GO:0021942 GO:0000160 GO:0008654 GO:0007031 GO:0007399 GO:0044267 GO:0006396 GO:0007162 GO:0007266 GO:0007229 ATP synthesis coupled proton transport tricarboxylic acid cycle vesicle-mediated transport fatty acid metabolic process cell redox homeostasis ATP biosynthetic process Glycolysis negative regulation of transcription ribosome biogenesis and assembly cellular process skeletal development protein complex assembly cerebral cortex radially oriented migration cell-cell adhesion involved in neuronal-glial interactions involved in cerebral cortex glial-mediated radial cell migration cerebellum structural organization radial glia guided migration of Purkinje cell two-component signal transduction system (phosphorelay) phospholipid biosynthetic process peroxisome organization and biogenesis nervous system development cellular protein metabolic process RNA processing negative regulation of cell adhesion Rho protein signal transduction integrin-mediated signaling pathway 44 39 95 63 46 40 56 68 96 85 27 52 5 5 5 5 31 41 20 96 26 75 11 38 36 MGP analysis was performed on GO BP categories which contains 4 ~ 99 genes (“Number of Probes”). The categories were sorted according to the integrated p value (p_int) and top 25 are represented. The categories related to energy cycle are noted (e.g., “proton transport”, “tricarboxylic acid cycle”, “ATP biosynthetic process”, and “Glycolysis”).