Fold changes were calculated as described previously using the 2-∆∆CT method [23] implemented in the DataAssist software version 3.0 (ABI), and significance was determined using one-way ANOVA in the R statistical package (version 2.13.2). Results and discussion Genes differentially expressed in mycelia and spherules Gene expression was assessed in a total of 12 samples derived from 4 replicate samples isolated from the following three growth phases: mycelia, day 2 spherules, and day 8 spherules. A photograph of mycelia and day 2 and

day 8 spherules grown in Converse medium is shown in Figure  1. The image shows the difference in shape and size between spherules and mycelia and the increase in spherule size between 2 and 8 days of culture. A custom oligonucleotide microarray (Nimblegen), which contained probes for all predicted ORFs of the RS strain of C. Pitavastatin purchase immitis was used to assess gene expression. 91% of the predicted ORFs were expressed Ruboxistaurin order in either mycelia or spherules, suggesting that the annotation and the detection of hybridization were

robust. Unsupervised clustering using the expression of all genes on the microarray revealed that mycelia samples clustered distinctly from spherule samples. Furthermore, spherule samples formed two sub-clusters based on the number of days in culture. A dendrogram showing that the four replicate samples cluster together is shown in Additional file 3: Figure S1. Fungal morphologic stage was the dominant determinant of the pattern of gene expression. Figure 1 Photomicrographs MRT67307 mw of C. immitis strain RS mycelium and spherules after 2 and 8 days of culture. Notice the large increase in size as the spherules mature. Genes that were significantly differentially expressed (p < 0.05) between the three conditions (mycelia, spherules on day 2 and 8) were identified in a supervised approach using a one-way ANOVA with appropriate corrections for multiple testing (see Methods). All the up- and downregulated genes differentially expressed between each of the three conditions

Exoribonuclease are detailed in Additional file 4: Table S2. A heatmap depicting expression levels in each sample for the top 100 differentially expressed genes is presented in Figure  2. The heatmap indicates there was limited variation in gene expression across the four replicates within each of the three conditions, suggesting that the data was highly reproducible. Multiple patterns of gene expression are evident comparing the three different conditions we studied. One cluster of genes was expressed to a lesser extent in the mycelia condition and a greater extent in both spherule conditions and another cluster of genes were expressed at a higher level in mycelia than in spherules. The expression of four genes (CIMG_08103, CIMG_09765, CIMG_10037, CIMG_10264) exhibiting the upregulated pattern was confirmed by RT-qPCR (see Figure  3 below).