coli rather than dual-species mixtures were used to study changes in transcription profile of E. coli due to cell separation. To this end, pure cultures of E. coli were processed using the same CAL-101 manufacturer procedure used for dual-species biofilm treatment, including cell dispersion and IMS. Differentially expressed genes were identified based on fold-change and statistical significance compared to the control (Figure 3) [24]. Only 10 and 45 of the 4,289 ORFs exhibited differential expression in two independent microarray studies

I and II, respectively (each microarray study was performed with two technical replicates of microarray slides and each microarray slide had three built-in replicates). A complete list of the differentially expressed genes is provided in Additional File 1: Full list of genes differentially Crenigacestat concentration expressed in sorted E. coli cells. Only eight of these genes showed consistent changes in both of the independent microarray studies (Table 1), with three genes up-regulated and five genes down-regulated in sorted E. coli cells in comparison

to unsorted E. coli cells. The selleck chemicals fold-change of gene expression ranged from 2.7 to -4.6 (Table 1). Differential expression of the eight genes in sorted and unsorted E. coli cells, as identified by the cDNA microarray analysis, was verified with qPCR using the 16S rRNA gene as a housekeeping gene. Seven out of the eight genes showed the same trend of differential expression (up-regulated or down-regulated in sorted cells) as revealed by the cDNA microarray analysis (Table 1). Moreover, the qPCR results indicated that five out of the eight genes exhibited less than two-fold change in sorted/unsorted cells. It suggested that the actual number of genes affected by the performance of IMS

sorting may be even less than eight. It further confirmed the effectiveness in preserving the transcriptome of E. coli cells by the method developed in this study. Figure 3 Plot of gene expression of Etomidate sorted/unsorted cells. Plot of one-sample T-test p-values with fold-change in gene expression for all ORFs in microarray study I. Vertical lines show the cutoff of fold-change of 2 (Log2 ratio of ± 1), while the horizontal line shows the cutoff of p-value 0.05. Genes located in the left-bottom corner (Log2 ratio <-1 and p-value <0.05) and in the right-bottom corner (Log2 ratio >1 and p-value <0.05) were considered to have their expressions changed due to dispersion/homogenization and IMS (immuno-magnetic separation) cell sorting. A total of ten genes were selected using these criteria, eight of which also differentially expressed in the independent microarray study II. Table 1 Genes identified as differentially expressed# between IMS sorted E. coli cells versus unsorted E.