RNA was extracted from a pool of three spleens taken from three month old nonobese diabetic (NOD) derived NOD.B6 Idd3 R450 congenic mice kept in sterile, specific pathogen free conditions. Target cRNA was prepared and hybridized to Eos custom GeneChip arrays (GPL24) as described for standard, commercially available Affymetrix GeneChips (Mahadevappa and Warrington 1999) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Data for each GeneChip was normalized using a proprietary method developed at Eos (Ghandour and Glynne 2000). Briefly, for each probe array in the series background subtracted average cell intensities were fitted to a gamma distribution. These normalized cell intensities were then used to calculate an average intensity (AI) for each probe set. The AI was calculated as the trimean (T) of the probes making up a given probe set (Tukey 1977). These AI values were subjected to a second round of normalization by setting the 70th and 90th percentiles equal to the same value for each array in the series.
