Global gene expression profiling in Escherichia coli K12 - The effects of integration host factor

We have used nylon membranes spotted in duplicate with full-length polymera se chain reaction-generated products of each of the 4,290 predicted Escheri chia coli K12 open reading frames (ORFs) to measure the gene expression pro files in otherwise isogenic integration host factor IHF+ and IHF- strains. Our results demonstrate that random hexamer rather than 3' ORF-specific pri ming of cDNA probe synthesis is required for accurate measurement of gene e xpression levels in bacteria. This is explained by the fact that the curren tly available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing deg radation rates (steady-state levels) are observed for the 25-base pair regi on of each message complementary to each ORF-specific primer. To evaluate t he DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical m ethods allowed us to identify and appropriately correct for experimental va riables that affect the reproducibility and accuracy of DNA microarray meas urements and allowed us to determine the statistical significance of gene e xpression differences between our IRF+ and IHF- strains. Our results demons trate that small differences in gene expression levels can be accurately me asured and that the significance of differential gene expression measuremen ts cannot be assessed simply by the magnitude of the fold difference. Our s tatistical criteria, supported by excellent agreement between previously de termined effects of IHF on gene expression and the results reported here, h ave allowed us to identify new genes regulated by IHF with a high degree of confidence.