genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Kidney Gene Expression Pattern in an in vivo Infection Model
In the search for specific differences in infection with S. aureus RN1HG ΔsigB vs. infection with
RN1HG, the same comparison was performed for the biological replicates separately (for
comparison see Fig. R.2.3; and Fig. R.2.4, panel ). This analysis resulted in 7 or 6
sequences/genes which were differentially expressed in the first biological replicate BR1 or the
second biological replicate BR2, respectively (Table R.2.1). Both lists did not display any overlap
indicating that the few regulated genes were not reproducible in the biological replicates,
although statistically significant (Fig. R.2.5 B).
Additionally, the statistical significance in the biological replicate BR1 was again caused by one
single sample, i. e. by one specific animal, which provided an outlier value to the data set
(Fig. R.2.5 C). The same sample accounted for the outlier in the values of the single gene
significantly regulated when combining the biological replicates (Fig. R.2.5 A).
The statistical significance of the 6 differentially expressed genes in the second biological
replicate was not caused by outliers (Fig. R.2.5 D), but the fold change in this comparison was
only moderate ranging from to -1.34 to -2.18 (mean: -1.72; median: -1.67).
In conclusion, the study could not provide any hints for statistically significant differences in
the expression pattern in murine kidney upon infection with S. aureus RN1HG or its isogenic sigB
mutant.
When asking for the reproducibility of the same treatment in both biological replicates in the
light of a possible influence on the detection of differential gene expression between the groups
infected with the two different S. aureus strains (for comparison see Fig. R.2.3; and Fig. R.2.4,
panel ), 33 sequences (i. e. 26 genes) were significantly different between the replicates of
infection with RN1HG, and 62 sequences (i. e. 31 genes) significantly differed between the
replicates of infection with RN1HG ΔsigB (Table R.2.1).
Of these genes, only a small fraction of approximately 20 % possessed an absolute fold change
greater than 2. For these few genes a high expression variation within one biological replicate
was observed or the statistical difference even was again due to one outlier array. Additionally,
most genes which differed between the replicates did not display any overlap with the genes
which were significantly different between the groups infected with the two different S. aureus
strains. Only some genes were statistically differentially expressed between the equally treated
replicates as well as between infection with RN1HG and RN1HG ΔsigB in the first biological
replicate. But as the statistical significance between samples infected with the two strains was
only caused by one single outlier value (Fig. R.2.5 C), it was clear that the small difference
between biological replicates did not prevent the identification of differential gene expression in
the comparison of host reaction to the two infecting S. aureus strains.
In summary, the comparison of same treatment groups in different biological replicates
revealed minor, negligible differences that did not influence the detection of differential
regulation when comparing the groups of infection with different S. aureus strains. The identical
reaction to infection with S. aureus RN1HG and S. aureus RN1HG ΔsigB was reliably measured in
the array study and the detection of differences was not prevented by biological variation
between the two independent infection experiments.
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