Differentially expressed genes were considered to be statistically significant if an absolute relative ratio was greater than 1.5 fold with an adjusted P value of less than 0.01. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE17942 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17942. GSK458 molecular weight Validation of microarray data by qRT-PCR Twelve differentially expressed genes with varying degrees of up- and down-regulation were selected from the microarray results for qRT-PCR. Primers for real-time RT-PCR were designed using Primer Express software
(ABI, Foster City, CA) [Additional file 3]. Each RT reaction mixture contained 5 μg of total RNA, 7.5 μg of random hexamers, 300 units of Superscript III reverse transcriptase (Invitrogen), 1 mM dNTP mix (1 mM each dATP, dGTP, dCTP, and dTTP), 10 mM DTT, and 20 units rRNasin® RNase inhibitor (Promega, Madison, WI). Samples were incubated
at 42°C for 2.5 h then at 70°C for 15 min. The synthesized cDNA was diluted 1/50 to 1/100 prior to use in real-time PCR. Real-time PCR reaction mixtures each contained 2.5 μL of cDNA, gene-specific primers at a final LY294002 purchase concentration of 100 nM each, and 10 μL of SYBR Green PCR master mix (ABI) in a total volume of 20 μL. Real-time PCR was carried out using a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany). Reactions were performed in triplicate.
A standard curve for each gene was constructed using known concentrations of L. interrogans serovar check details Copenhageni genomic DNA. The gene encoding flagella subunit B, flaB, was used to normalize all data. Melting curve analysis confirmed that all PCRs amplified a single product. Acknowledgements This work was supported by grants from the Australian Research Council and the National Health and Medical mafosfamide Research Council. KP was supported financially by the Faculty of Medicine, Chulalongkorn University, Thailand. KP also acknowledges with thanks the kind help from her colleagues at the Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Thailand during her absence. Electronic supplementary material Additional file 1: Table S1. List of genes upregulated in serum, with an adjusted P value of < 0.01. (XLS 24 KB) Additional file 2: Table S2. List of genes downregulated in serum, with an adjusted P value of < 0.01. (XLS 34 KB) Additional file 3: Figure S1. Comparison of quantitative RT-PCR and microarray data for twelve genes with varying degrees of up- and down-regulation selected at random. (DOC 36 KB) Additional file 4: Table S3. Sequences of primers used for PCR and for real-time qRT-PCR to confirm microarray data for some genes. (DOC 24 KB) References 1. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM: Leptospirosis: a zoonotic disease of global importance.