BMC Genomics 2009, 10(Suppl 2):S4 http://www.biomedcentral.com/1471-2164/10/S2/S4 Table 4: Effects of sub-MIC of antibiotics on P. multocida putative virulence factors * Ratio of treatment/control normalized intensities. ns: no significant change in gene expression with sub-MIC of antibiotic. GeneID 1244083 1244119 1244120 1244121 1244128 1244133 1244195 1245051 1245061 Product Name Hypothetical protein PM0736 PhyB PhyA HyaE HexA Hypothetical protein PM0786 Hypothetical protein PM0848 ClpB Hypothetical protein PM1714 multocida strain Pm70 was constructed by NimbleGen Systems. The array design included 23 perfect match (PM) and mismatch (MM) probes per target gene, and each probe was spotted twice. Reverse transcription, cDNA labeling, and hybridization were conducted by Nimble- Gen Sytems according to their established protocols. Briefly, three independent RNA samples (10 g each) from untreated and antibiotic treated Pm70 were reverse transcribed using Random Hexamer (Gibco) and Super- Script II Reverse Transcriptase (Invitrogen). Biotin end- labeling of cDNA utilized Terminal Deoxynucleotidyl Transferase (Promega) with Biotin-N6-ddATP (Perkin Elmer); hybridization and washes were conducted using NimbleGen Systems protocol. Detection of hybridized biotin-labeled probe used Fluorolink Cy3 Labeled Streptavidin (Amersham Pharmacia). Raw intensity data (single channel) were log transformed, and normalization was done by quantile normalization [28] and robust multichip analysis (RMA) algorithm [29]. The experimental design and all microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE12779). ANOVA followed by step-down Bonferroni correction for multiple testing was conducted to identify genes with significant changes in expression relative to untreated control (p  0.05) in any group. Sig- nificant changes between control vs specific antibiotic treatments were identified by performing Dunnett's test [30]. The differences in gene expression in response to antibiotics were calculated as the ratio of treatment vs control intensities. Real time RT-PCR The majority of significant changes in gene expression identified in this study were less than one-fold. Therefore, to validate changes in gene expression, we performed duplex real time quantitative RT-PCR (qPCR) of six genes: gyrb, impA, rpsg, hpkR, recQ, and murA using gapdh as the internal standard. The RNA template used for qPCR was the same RNA that was used for microarray hybridiza- Locus DnaK phyB phyA hyaE hexA OmpA tadB clpB - AMX* ns ns -0.80 -0.64 -0.74 -0.83 -0.51 -0.66 -0.74 CTC* -0.74 -0.84 -0.71 -0.91 -0.69 -0.75 ns ns -0.91 ENR* 1.50 1.41 1.71 1.27 -0.73 ns ns 2.14 ns tions. The probes and primers for duplex RT-PCR were designed using Beacon Designer software (Additional file 4, supplemental table 3). Reactions were performed using the Platinum® SYBR® Green One-Step qRT-PCR Kit (Invit- rogen Corporation, Carlsbad, CA). Amplification and detection of specific products were done using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laborato- ries, Inc., Hercules, CA). Regression analysis of the Ct val- ues of the test RT-PCRs was used to generate standard curves. The mean Ct value for the GAPDH mRNA-specific reactions was used to normalize the test values. Regres- sion analysis of the gene expression trends determined by qPCR and microarray analysis was performed in Microsoft Excel. Functional analysis of differentially expressed genes Initial functional analysis of genes with significant changes in expression was done using clusters of ortholo- gous groups (COG) classification [31]. For each COG cat- egory in the genome, the percent increase or decrease in expression of genes belonging to that category for each antibiotic treatment was calculated. The COG functional categories described for P. multocida genes [6] along with the description for proteins encoded by the genes at Uni- prot [32] were used to identify overall themes in antibiotic response. Specific functions of P. multocida proteins were deduced from information available for E. coli orthologs at the EcoCyc database [33]. Network analysis of DE genes was done in Pathway Studio (Ariadne Genomics) as described earlier [11]. Briefly, we built interaction net- works in Pathway Studio with proteins of interest includ- ing the upstream regulators and downstream targets. In the interaction networks, different colors were used for nodes to indicate significant increase (red), decrease (green), or no change (pink) in gene expression in response to sub-MIC of antibiotic. Entities in the interac- tion map that were not present in the Pm70 genome were shown in gray color. Page 8 of 10 (page number not for citation purposes)