Where multiple probe sets for a given gene were present on the array, these were summarized using the maximal expression value.
To rigorously compare the expression profiles of the major clusters, the scaled and normalized hybridization intensities were retrieved for each gene and plotted by group as a percentage of each gene's maximal expression value during the time course (because of the limited number of probe sets in the Biphasic group, these were excluded from subsequent analysis).
Specifically, when the putative targets of NF-κB were sorted in descending order according to their maximal expression value in MmBMM (over all time points), the top 10% genes were significantly enriched for the NF-κB+ISRE pair (p < 0.005; see Methods).
This strategy therefore revealed only the most strongly stage-specifically regulated genes; maximal differences of RNA expression values of 54-fold (ORF TGME49_295662, merozoite-specific) and 404-fold (ORF TGME49_215980, tachyzoite-specific) were measured.
Even so, use of the maximal expression as the control value allows a comparison of the expression dynamics of viral genes both at individual time points and across the entire infection period.
Therefore we also determined the median of the highest expression value of each gene, representing the maximal expression potential using the same threshold value of 200.
One group extended a model that explored the effects of preconditioning on the responses of Toll-like receptors to lipopolysaccharides (Riviere et al., 2009) by using a different parameter value for the maximal expression of the Toll-like receptors, based on published research that described the effect of overexpression of Toll-like receptors (Bihl et al., 2003).
The first group consists of 738 genes, or almost a fifth of the genome, that have a low expression level (with the maximal expression being below 5x) and an average CV value of 0.38.
To compare developmental gene expression profiles, published time series of high-throughput RNA expression data for DD and DP8 were expressed as percentage of maximal expression and stage-matched to new RNA expression data for DL, PP and DF.
Genes represented by multiple probesets were averaged, and expression values were scaled to the probeset with the maximal variance for that gene.
