Calculation of the relative quantification of the target genes was done using the Comparative CT (ΔΔCT) method [39]. The protocol of the PCR is given as described below: Each 20-μl PCR reaction mixture contained 2 × Power SYBR Green PCR Master Mix (Applied Biosystems, Streetsville), 100 nM of each of forward and reverse primer, and 5 μl of template cDNA. Synthesis of the template cDNA was carried out in a 20-μl reaction mixture containing 500 ng RNA, using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), buy Vorinostat which contains random primers for the synthesis of cDNA. The real-time PCR thermal profile included the heat-activation of AmpliTaq Gold DNA Polymerase at 95°C for 10 min,

40 cycles of denaturation at 95°C for 15 s, and primer annealing and see more extension at 60°C for 1 min. The PCR reactions were carried out in 96-well plates using a StepOnePlus thermocycler (Applied Biosystems, Streetsville, ON, Canada). The primers used in the real-time PCR are given in Table 10. Table 10 Oligonucleotide primers used in the real-time PCR Gene Forward primer Reverse primer dmsA ATGTTGCCGGACAAGCACAAGATG TCTCAATGGACAACGGCTACCACA dmsB Z-DEVD-FMK purchase AACAGGCATCGATTGCACCGTTAC

ACTTGGACGTGCGTGTTTATTGGC napB GCGCATGGCAACCTAAACATTGGT TACAGGCTTTGCAGTAGCGGAAAC napD TCGGCTAAAGCAAGCTGTCTGTCA TAGCGCAAGTGAAAGCGGACATTC napF ACAACCGTCTCCGCAACTTCTACA TTGGCTACAACGGAAGAAGCATGG ilvH GAAAGTTTAACCGTTGCGCCGACT ACGTTCAATATGCTCGGTAGGGCT pgaA GGGAACCGGTGTGAATGCAATGAA TGTTGGAACGTTTGTGAAGACGCC pgaC ATCGTTGCGTTACACCAAGCGAAC ACCGACATACTTGCCTCTTGCGAT apxIVA TTGGACTTCACCTGCAAACATGCC

CGGGCAAATATTCCAAAGCGCAGA relA TCGGACAGTTGAAGTGGGAAT TGCAAGGCGATTACTCGGTAA Oxymatrine syp AAGAAACGCCGAATGATGCACAGG ACACCTCGATAGCACCACCTTTGT lamB CTGCTAAAGAGAGTTTACCGATGCCA TGCAACATTACGGGCAGGTAAACG malK GCGTGTTGCAATTGGACGTACCTT CATGGCTTCGATTTGGTCATGCGT malM AGCGACACCGTCAAAGACAGAACT CCAACGTTTGGCTAAATGTGCGGA malT TCCTTGATGAGCTTTCGACCCACA TAAACCGAGCACCTGCCATTCTCT malP ACGCTTAGCCGCCTGCTATTTAGA CACGCATCGCCTTCTTCATGTTGT malQ ATGCCTATCGGCCTTTACCGTGAT ACCGACAGAGGCATCTAGCACAAA malE AACCGATGAAGGACTCACAACCGT TTTCCGCATTCGCCATAGTTGCTG malF TGCCGTTAATGATTGCCAGCTTCG GCAGCCGCTAAACCAAAGTCTTGT malG AGTGTTACTCATGCGGACGGAAGT GCATACGCAGCAGTGGTTGAAAGT Acknowledgements This work was supported by the grants from the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Agriculture, Food, and Rural Affairs, Canada. We thank Drs. Jeff Caswell and Andrew Brooks for providing us with bronchoalveolar lavage fluid, and Jing Zhang and Devon Metcalf for their help with real-time PCR experiments. Electronic supplementary material Additional file 1: Differentially expressed genes of the BALF-exposed A. pleuropneumoniae malT mutant, grouped according to biological role. Analyzed microarray data of the BALF-exposed A. pleuropneumoniae malT mutant. (DOC 274 KB) References 1. Rycroft AN, Garside LH:Actinobacillus species and their role in animal disease.