All samples had a RNA integrity number greater than 7. Microarray design and hybridization Known and predicted ORFs from the C. immitis genome (RS strain) were previously identified using sequence data available at the Broad Institute [14]. This information was supplied to Roche Nimblegen in order to manufacture a custom oligonucleotide array consisting of 68,927 probes (Nimblegen custom array OID30589). Probes were 60 nucleotides in length and the expression of the majority of
genes was assayed using 7 different probes printed in duplicate. The expression of small genes was assayed with fewer probes. Twelve custom microarrays fit on a single slide such that all the samples in this study (4 × mycelia,
4 × day 2 spherule, and #Adriamycin research buy randurls[1|1|,|CHEM1|]# 4 × day 8 spherule) could be assayed for gene expression in a single experiment to eliminate technical batch effects. Ten μg of total RNA at a concentration greater than 1 μg/ml from each sample was used for microarray hybridization. Total RNA was converted to cDNA, labeled with dye, and hybridized to the microarray by the VA San Diego Gene Chip Microarray Core according to the Nimblegen protocol. All C. immitis genes are referred to by their locus tag and further information about these genes can be found at the Coccidioides group database at the Broad Institute http://www.broadinstitute.org/annotation/genome/coccidioides_group/MultiHome.html. FungiDB (http://fungidb.org/fungidb/) was also used for annotation because it has see more more informative gene names for many genes. Microarray data analysis Quality control analysis and normalization of microarray gene expression data were performed as previously described [15]. Briefly, several quality control assessments (e.g., boxplots
and volcano plots) were applied to assess microarray data quality. Unsupervised clustering was also performed using the web-based tool ANAIS [16] to determine if samples clustered as expected based on the expression of genes in each sample. All arrays passed quality control filters and no outliers were found. Differentially expressed probes were identified between mycelium, day Guanylate cyclase 2C 2 spherule and day 8 spherule conditions using a one-way ANOVA and the Tukey post hoc test implemented in GeneSpring GX version 11.5 (Agilent Technologies Inc.). The false discovery rate (FDR) associated with multiple tests was corrected for using the Benjamini-Hochberg method [17]. In a conservative approach, a gene was only identified as differentially expressed if all probes for that gene had a fold change greater than 2 or less than −2 and an ANOVA p-value (Tukey and FDR corrected) less than 0.05. Fold changes were calculated for each gene that passed this filter by averaging across the seven probes.