Gene expression analysis in intestinal epithelial cells of germ-free versus wildtype mice

Gene expression was analyzed in intestinal epithelial cells of germ-free and wildtype mice. Ileums and proximal colons of 4 C57/Bl6 control and 4 C57/BL6 germ-free mice were collected and embedded in OCT. Tissue sectioned (10 mikroM) and laser capture microscopy was performed using a Zeiss/Palm laser capture system. Total RNA was extracted from about 16000 cells of the catapulted samples using RNA microprep kit (Stratagene). These 16 000 cells derive from all 4 animals (pooled approx. 4 000 cells from each mouse). We retrieved between 50 and 150 ng total RNA. Fourty ng total RNA was expanded by two rounds of in vitro cRNA amplification and affymetrix genechip Moe403 were probed at the Swegene Resource Center Lund, Sweden. Array data analysis was performed using dCHIP2004 software.