cRNA 15g was fragmented and hybridised to Mu11KsubA and Mu11KsubB arrays. Hybridised arrays were washed and stained with Streptavidin R phycoerythrin using the GeneChip Fluidics Station 400 and scanned with a Hewlett Packard GeneArray Scanner according to the manufacturers proto cols. All array images were visually inspected for defects and quality. Arrays with excessive background, low signal intensity or major defects within the array were eliminated from further analysis. GeneChip MAS 5. 0 software was used to evaluate the hybridisation intensity, compute the signal value for each probe set and make an absentpresent call. GeneChip signal data for the samples analysed in this study are available under Accession Number. Data IPI-145 normalisation and filtering GeneChips were required to pass standardised quality control criteria.
RNA quality was monitored by the ratio of frequencies measured by independent probe sets representing 5 and 3 regions of glyceraldehyde 3 phosphate dehydrogenase. This ratio must be more than 0. 4. Filtering criteria for individual probe selleck inhibitor sets required that a probe set was called present or a signal of 50 or more in at least one of the samples. All filtering criteria were passed by 6384 probe sets and were subject to the statistical analysis described below, and probe sets that did not meet these criteria were not included in subsequent analyses. Hierarchical clustering For hierarchical clustering of probe sets and arrays, the Log 2 scale MAS5 expression values from each probe set were first z normalised so each probe set had a mean expression level of zero and a standard deviation of one across all sam ples.
Then these normalised profiles were clustered hierarchi cally using an unweighted paired group method with arithmetic mean, and the Euclidean distance measure. Identification of genes associated with lupus nephritis and response to sirolimus therapy The disease related fold change differences were calculated by determining the difference in the log 2 signal of the 12 week old asymptomatic mice and the combined 36 and 42 week old diseased mice. Analysis of variance was performed using this metric to identify disease related differ ences. Raw p values were adjusted for multiplicity of testing using the false discovery rate procedure of Reiner and colleagues using Spotfire. Genes with a FDR p 0. 05 and an absolute fold change of two of more in the comparison between disease and asymptomatic groups were identified as being significantly associated with lupus nephritis. Lupus nephritis genes were identified as being sig nificantly associated with response to sirolimus treatment if they met an FDR p 0. 05, in comparison between sirolimus treated and disease groups.