Figure 7

Validation of FPKM-based gene expression by quantitative RT-PCR. (A) Identification of genes with minimal variation in expression between strains. Gene expression of candidate normalization genes (TEF1, TEF3, RPS1B, TDH1, ACT1, and CBP1) was determined by qRT-PCR. Expression values were normalized to one member of the gene set and the total difference in cycle threshold between strains (y-axis) determined for the remaining genes. Colors correspond to which gene was used for normalization and the resultant total variation in cycle thresholds. The process was repeated iteratively by removing the gene, which when used as the normalizer, resulted in the greatest overall difference in expression between strains. Included genes are listed under the x-axis. Horizontal bars represent averages. Significantly different total cycle threshold variations determined by Student’s t-test are indicated (*, P < 0.5; **, P < 0.1; ns, non-significant). (B) Correlation between FPKM and qRT-PCR determination of gene expression between G186A and G217B yeasts. Data points represent the log₂-transformed value of the fold-change in expression determined by qRT-PCR after normalization to TEF3 (x-axis) or by FPKM ratio (y-axis). Data point color indicates genes with no difference in expression (black; < 5-fold), genes upregulated in G186A (green; > 5-fold), and genes upregulated in G217B (red, > 5-fold). Error bars represent the standard deviation of the relative expression of three replicate yeast cultures for each strain. (C) Relative expression of selected genes in G186A yeasts compared to G217B yeasts. Bars represent the average fold change (log₂) and error bars represent the standard deviation (n = 3 for each strain). Analyzed genes (x-axis) and data are colored to indicate genes with no enriched expression in either strain (black), genes upregulated in G186A yeasts (green; > 5-fold), and genes upregulated in G217B yeasts (red; > 5-fold). Asterisks denote genes with established roles in Histoplasma yeast virulence.