epidermidis strain RP62A, as
well as unique ORFs in S. epidermidis strain 12228. The GeneChips were composed of cDNA array containing PCR products of 2316 genes and oligonucleotide array containing 252 genes. Reverse transcription were performed using see more 2 μg of total RNA using T7 promoter learn more primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA), and then cRNA was transcribed from the resulting cDNA as template. cRNA prepared form 1457ΔlytSR and the parent strain was labelled using the dyes Cy3 and Cy5 according to the manufacturer’s instructions(Amersham, Piscataway, New Jersey) respectively. Microarray hybridization (at 42 °C for 16 h) and washing of the slides at 50 °C were performed according to the manufacturer’s instructions. Hybridized slides were scanned by Agilent Scanner (G2655AA) at a 10-μm resolution.
Data of each image were normalized to the mean ratio of means of all features. Mean values and standard deviations of gene expression ratios based on three spot replicates on each microarray were calculated in Microsoft Excel XP. The complete set of microarray data was deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO, available at http://www.ncbi.nlm.nih.gov/geo/ and is accessible through GEO Series accession number GSE20652. Validation of microarray data by Real time PCR To confirm the results of the microarray data, the relative expression levels of the lrgA, ebsB, STK38 arcA, serp2169 and leuC genes were determined by real-time PCR with gene-specific primers, designed according to the genomic PF-02341066 manufacturer sequence of S. epidermidis RP62A (GenBank accession number CP000029). The sequences of the primers are shown in Table 4. Briefly, DNase-treated RNA was reverse transcribed using M-MLV and a hexamer random primer mix. Appropriate concentration of cDNA sample was then used for real-time PCR using an ABI 7500 real-time PCR detection system, gene-specific primers, and the SYBR Green I mixture (Takara, Dalian, China). Relative expression levels were determined by comparison to the level of gyrB expression in the same cDNA preparations.
Statistical analysis Experimental data obtained were analyzed with the SPSS software and compared by Student’s t test. Differences with P < 0.05 were considered statistically significant. Acknowledgements We thank Dr. Patrice Francois (Genomic Research Laboratory, University of Geneva Hospitals, Switzerland) for repeating the microarray experiments. This work was supported by the 11th Five-Year Plan of the Ministry of Sciences and Technology (2010DFA32100, 2009ZX09303-005, 2008ZX10003-016), the Hi-Tech Program of China (863) (2006AA02A253), the Scientific Technology Development Foundation of Shanghai (08JC1401600, 10410700600), National Natural Science Foundation of China (30800036), the Research Initiation Grant for Young Faculty of Fudan University (09FQ43).