Each experimental group consisted of 10 mice per group, except the control group of germ-free mice, which consisted of 5 mice per group. After an acclimatization period of at least 4 weeks, feces were freshly collected from the conventional mice. Feces from the same group was pooled and mixed in PBS. Next, 200 μl of 100 mg/ml of this mixture was given by oral gavage to germ-free mice of 12-14 weeks old. After transfer, recipient mice were individually housed in IVC cages for another 4 weeks. Mice were then killed, and part of the terminal ileum was collected, snap-frozen and stored at -80 until gene expression profiling.
Growth protocol
Young (7-10 weeks) or old (17 months) C57BL/6JRccHsd conventional female mice were purchased from a commercial supplier (Envigo, Horst, the Netherlands). Female germ-free mice of 12-14 weeks were obtained from a breeding colony at the animal facility of Radboud University Nijmegen Medical Centre (Nijmegen, the Netherlands). All animals were put on an autoclaved Rat/mouse maintenance V153X R/M-H diet (Ssniff, Soest, Germany) directly after weaning in the case of germ-free mice, or directly after arrival in the case of conventional mice. The mice were kept on this diet throughout the experiment. Conventional mice were housed in IVC cages and germ-free mice were housed in germ-free isolators.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy micro kit from Qiagen. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
Purified total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.40.2).
