Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 Table 2 Bacterial strains and plasmids used in this study Strains and plasmids Characteristics Strains S. Enteritidis Wild-type clinical isolate PT4 PT4 PT4 containing helper plasmid (pKOBEGA) pKOBEGA, AmpR PT4ΔssaN PT4 ΔssaN::Km, in-frame deletion, KmR PT4ΔssaR PT4 ΔssaR::Km, in-frame deletion, KmR PT4ΔsspH2 PT4 ΔsspH2::Km, in-frame deletion, KmR E. coli MC4100 Used as template for amplication of kanamycin resistance gene Plasmid Vector for recombination experiments, pKOBEGA AmpR Source/ reference 34a This study This study This study This study Gift from J. M. Ghigo Gift from J. M.Ghigo a : S. Enteritidis strain was typed as PT4 and named as the strain KS8822/88 in this study. indicated. To determine the number of living intracellu- lar bacteria, the infected cells were scraped using a cell scraper and the amount of living host cells was counted under a microscope after staining with Trypan blue. Host cells were lysed with 1% Triton X-100 in 1 × phos- phate-buffered saline (PBS) at room temperature for 5- 10 min, 20 μl of the lysate in ten-fold serial dilutions in PBS were added to LB agar plates, and the numbers of bacteria were reported as CFU (colony forming units). RNA extraction Two methods were used to extract bacterial RNA from infected cells. At 1 or 8 h post infection, the intracellular bacteria were recovered from infected macrophages and lysed with 1% Triton X-100 [34]. The lysate was first cen- trifuged at 250 × g at 4°C to remove pieces of broken host cells and then the supernatant was centrifuged at 12,000 × g for 5 min to precipitate bacterial cells. The approach developed for recovering bacteria from infected cells ensured the minimum contamination of extracted bacterial RNA with eukaryotic RNA. Otherwise, infected macrophages were lysed in 0.1% SDS, 1% acidic phenol, 19% ethanol in water on ice for 30 min [60] and similar results were obtained. Total bacterial RNA was extracted and purified using the Promega SV total RNA purifica- tion kit according to the manufacturer’s instructions (Promega). Control RNAs from bacteria grown in LB broth to mid-logarithmic phase and from eukaryotic cells were isolated using the same RNA purification kit. RNA integrity was monitored by agarose gel electrophoresis and using the Agilent 2100 system (Agilent). The con- centration and purity of RNA was measured by spectro- photometry Bio-RAD SmartSpee™3000 (Bio-Rad). Microarray hybridisation The ‘SALSA’ Salmonella serovar microarray was devel- oped with 5080 Salmonella genes from different Page 9 of 13 serovars of Salmonella including 196 S. Enteritidis PT4 specific genes. More details are available at http://www. ifr.bbsrc.ac.uk/safety/microarrays/#protocols. A type II experiment design was used for hybridisation with labelled bacterial genomic DNA as a reference channel in each experiment [70]. The protocol for hybridisation was as previously described [52]. Briefly, total bacterial RNA (5 μg) from Salmonella grown in host cells or in LB broth was converted into cDNA using Superscript II reverse transcriptase and random primer as recom- mended by the manufacturer. The cDNA produced from the reverse transcription reaction and genomic DNA was subsequently fluorescently labelled using Cy5 or Cy3 dyes by random priming, with increasing label- ling efficiency using Klenow enzyme. Labelled cDNA and genomic DNA were hybridized with the microarray slides overnight at 65°C. After hybridization, the slides were carefully washed, dried and scanned. All hybridiza- tions were performed with at least three biological replicates. Microarray data analysis Fluorescent intensity data from each array were col- lected using a GenePix 4000A scanner (Axon Instru- ments). To compensate for unequal dye incorporation, data were centred by bringing the median natural loga- rithm of the ratios for each group of spots printed by the same pin to zero. The data were analyzed using GENE-SPRING™6 software (Silicon Genetics). The sig- nificance of the centred data at P = 0.05 was determined using a parametric-based statistical test, adjusting the individual P-value by the Benjamini and Hochberg false discovery rate multiple test correction. Microarray data accession number The supporting microarray data have been deposited in the Array Express database http://www.ebi.ac.uk/ arrayexpress with accession number E-MEXP-1438. Quantitative real time RT-PCR Primer3 software http://frodo.wi.mit.edu[71] was used to design the primers for quantitative real time RT-PCR and purchased from Sigma (Table 3). The primers were designed to have similar melting temperatures and lengths of PCR products. About 1 μg of total RNA from the bacteria after 2 or 8 h infection of U937 cells or growth in LB medium was reverse-transcribed to 1st strand cDNA using AMV reverse transcriptase and the random primer provided in the kit following the manu- facturer’s instructions (1st Strand cDNA Synthesis Kit for RT-PCR (AMV)+, Roche). The resultant cDNA was used as a template during PCR, which was performed on a Roche LightCycler instrument and using the Light- Cycler FastStart DNA Master SYBR green I kit (Roche).