Yamamoto et al. BMC Genomics 2012, 13:335 http://www.biomedcentral.com/1471-2164/13/335 meta-analysis technique, accumulates statistical evidence from a set of genes in order to build a more powerful test than can be achieved by analyzing individual genes. In the present study, we first predefined the group of genes for each GO term: all 3 categories, i.e., BP, cellular component (CC), and molecular function (MF), were used. More than 20,000 gene sets were annotated by GO terms. We also used gene sets defined by pathways in the TRANSPATH database. To obtain the p-values for individual genes, Welch’s t test was performed. The indi- vidual p-values of the genes included in the gene set were integrated to obtain the integrated p-value for the gene set, as described previously (http://metagp.ism.ac. jp/). A gene set containing too small a number of genes is, in principle, unsuitable for evaluation of overrepre- sented gene sets. Preliminary examination suggested that GO terms containing more than 100 genes provided relatively little information because these terms repre- sent too broad a concept to give a foothold for further biological investigation. Therefore MGP analysis was applied to gene sets consisting of 4–99 genes. Quantitative real time RT-PCR For RT-PCR, total RNA was extracted as described above (n = 6 per group). The cDNA samples were synthesized using an Improm-IITM Reverse Transcript- ase kit (Promega Corporation, Madison, Wl) according to the manufacturer’s instructions. Briefly, 5 μl of RNA (50 ng) and primer were added to 15 μl of reverse tran- scription reaction mix (Improm-IITM Reverse Tran- scriptase system). Annealing was performed by placing the tubes in a controlled-temperature heat block equili- brated at 25 °C and incubated for 5 minutes. Extension was performed in a controlled-temperature heat block at 42 °C for up to 1 hour. The extension temperature was optimized between 37 °C and 55 °C. Real time RT-PCR was performed using the TaqManW Gold RT-PCR Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. An ABI Prism 7900HT (Ap- plied Biosystems) was used with the following thermal cycling conditions: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, 40 cycles each at 95 °C for 15 sec and 60 °C for 1 min. Data were normalized against Irf1 [36,37]. Statistical analyses PCR data were calculated as the mean values and are represented in Figure 10 by columns indicating the rela- tive value of each group, which was normalized to the maximum value in the 4 groups. Differences in mean values among groups were analyzed by Dunnet test and were considered significant at p < 0.05. The statistical methods used for the microarray analysis are described above. Page 14 of 15 Abbreviations 0WexGF: Germ-free mice with bacterial reconstitution at the time of delivery; 5WexGF: Germ-free mice with bacterial reconstitution at 5 weeks of age; BP: Biological Process; CC: Cellular Component; GF: Germ-free; GO: Gene Ontology; GSEA: Gene set enrichment analysis; IFN: Interferon; IL: Interleukin; LI: Large intestine; MF: Molecular Function; MGP: MetaGene Profiler; SI: Small intestine; SPF: Specific pathogen free; Th: T helper; TLR: Toll like receptor; Treg: Regulatory T. Competing interests The authors declare that they have no competing interests. Acknowledgements This work was supported by Keio University School of Medicine, Central Institute for Experimental Animals and Tsumura Research Laboratories. Author details 1Center for Kampo Medicine, Keio University School of Medicine, 35 Shinano- machi, Shinjuku-ku, Tokyo 160-8582, Japan. 2Tsumura Research Laboratories, Tsumura & Co., Ami, Ibaraki 300-1192, Japan. 3Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan. 4Central Institute for Experimental Animals, Kawasaki, Kanagawa 216-0001, Japan. Authors’ contributions MY participated in the design of the study, data analysis, coordination, and drafted the manuscript. RY carried out microarray data analysis including MGP. KM participated in animal experiments. MiN and KT carried out RT-PCR. AI, KH and YO participated in the design and coordination of the microflora reconstitution experiments. MaN, SI, and SM participated in the design of statistical and bioinformatics analyses. KW conceived the study and participated in its design and coordination. All authors read and approved the final manuscript. 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