mRNA transcription by RT-qPCR for selected pneumococcal genes.

Using the random stratification method, differentially expressed genes were stratified and genes then randomly selected from each stratum for validation. Both up- and down-regulated genes lists were divided into strata containing three genes and random number generating software (Apple Inc.) was used to select the gene for validation. The data are from five independent infection experiments and three control samples, i.e. pneumococci grown without adherence to cells. RT-qPCR was done in triplicate and fold expression changes for each gene by microarray analysis are shown alongside. To demonstrate that possible contamination with host RNA did not impact on pneumococcal RT-qPCR mRNA expression data, as a control, RT-qPCR was also done using Met-5A PMC uninfected cDNA; for all genes examined, the level of non-specific amplification of Met-5A cell cDNA was * denotes statistical significance (Ct values) mRNA transcription by RT-qPCR for selected pneumococcal genes.