The MRSA SA1450/94 is a member of the Iberian clone (ST 247). Source: German reference centre for staphylococci, Wernigerode
Growth protocol
S. aureus was cultured in brain heart infusion (BHI) medium (Becton Dickinson GmbH, Heidelberg, Germany) at 37°C with aeration. An overnight culture was diluted 100fold in fresh BHI broth and further incubated to an OD600 of 0.8-1.0 to ensure exponential growth conditions.
Extracted molecule
total RNA
Extraction protocol
Culture aliquots of 10 ml were stabilized by incubation with two volumes RNAprotect Bacteria Reagent (QIAGEN, Hilden, Germany) for 5 minutes at room temperature. Subsequently, cells were harvested by centrifugation, lysed in presence of 400 µg/ml lysostaphin (Sigma-Aldrich, Taufkirchen, Germany) and total RNA was isolated using the RNeasy Midi Kit (QIAGEN) following the manufacturer’s instructions. RNA concentration and integrity was determined by photometric measurement (Nanodrop, Nanodrop Technologies Inc., Wilmington, USA) and agarose gel electrophoresis.
Label
Cy3
Label protocol
Total RNA (6 µg) was transcribed into cDNA using the Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions. For direct fluorescent labeling of the cDNA, the transcription reaction was performed in a total volume of 40 µl in presence of 0.1 mM cyanine-3’ or cyanine-5’ labeled dCTP (Perkin Elmer Life Science, Mechelen, Belgium) in addition to 0.2 mM dCTP, 0.5 mM dATP, 0.5 mM dGTP and 0.5 mM TTP, 75 µg/ml random hexamer primers (Amersham, Bioscience, Freiburg, Germany) and 25 U/ml RNase-Out (Invitrogen). RNA was degraded by alkaline hydrolysis at 65°C and cDNA was purified using the MinElute PCR Purification Kit (QIAGEN).
Subculturing of the clinical methicillin-resistant S. aureus (MRSA) SA137/93A displaying heterogeneous intermediate vancomycin resistance (MIC: 8 mg/L in BHI) in presence of 6 mg/L vancomycin generated a mutant with homogeneous intermediate vancomycin resistance, that showed an MIC value of 16 mg/L in BHI and was designated SA137/93G. Source: Reipert, A., K. Ehlert, T. Kast, and G. Bierbaum. 2003. Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin. Antimicrob.Agents Chemother. 47:568-576.
Growth protocol
S. aureus was cultured in brain heart infusion (BHI) medium (Becton Dickinson GmbH, Heidelberg, Germany) at 37°C with aeration. An overnight culture was diluted 100fold in fresh BHI broth and further incubated to an OD600 of 0.8-1.0 to ensure exponential growth conditions.
Extracted molecule
total RNA
Extraction protocol
Culture aliquots of 10 ml were stabilized by incubation with two volumes RNAprotect Bacteria Reagent (QIAGEN, Hilden, Germany) for 5 minutes at room temperature. Subsequently, cells were harvested by centrifugation, lysed in presence of 400 µg/ml lysostaphin (Sigma-Aldrich, Taufkirchen, Germany) and total RNA was isolated using the RNeasy Midi Kit (QIAGEN) following the manufacturer’s instructions. RNA concentration and integrity was determined by photometric measurement (Nanodrop, Nanodrop Technologies Inc., Wilmington, USA) and agarose gel electrophoresis.
Label
Cy5
Label protocol
Total RNA (6 µg) was transcribed into cDNA using the Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions. For direct fluorescent labeling of the cDNA, the transcription reaction was performed in a total volume of 40 µl in presence of 0.1 mM cyanine-3’ or cyanine-5’ labeled dCTP (Perkin Elmer Life Science, Mechelen, Belgium) in addition to 0.2 mM dCTP, 0.5 mM dATP, 0.5 mM dGTP and 0.5 mM TTP, 75 µg/ml random hexamer primers (Amersham, Bioscience, Freiburg, Germany) and 25 U/ml RNase-Out (Invitrogen). RNA was degraded by alkaline hydrolysis at 65°C and cDNA was purified using the MinElute PCR Purification Kit (QIAGEN).
Hybridization protocol
Hybridization was done with equal amounts of cDNA probes displaying similar picomoles of incorporated dye. Fluorescence-labeled cDNA probes were mixed in hybridization buffer (Scienion) in a total volume of 55 µl and denatured at 95°C for 2 min and subsequently applied to the microarray slide followed by incubation at 42°C for 72 hours under humidified conditions according to the manufacturer’s instructions. Hybridized microarrays were washed at room temperature in SSC buffer with decreasing salt concentrations (1 x SSC / 0.3% SDS for 5 min, 0.2 x SSC for 5 min, 0.06 x SSC for 30s).
Scan protocol
The intensity of fluorescence of the microarray was scanned with a GenePix 4000B scanner (Axon Instruments/Distribution by Biozyme Scientific GmbH, Hessisch Oldendorf, Germany). TIFF images were captured and analysed with GenePixPro4.1 software (Axon Instruments). The actual signal intensity was calculated by substracting the mean value of the local background intensity from the mean value of the signal intensity of the individual spot.
Description
SA1450/94_SA137/93G_Chip ID186
Data processing
The data sets were normalized by using Acuity 3.1 software (Axon Instruments) and by applying the LOWESS algorithm. Significant changes of gene expression were determined by implementing SAM (significance analysis of microarrays, http://www-stat.stanford.edu/~tibs/SAM/ (Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc.Natl.Acad.Sci.U.S.A 98:5116-5121.)) using the one class response and a false discovery rate of < 1% with a medium number of falsely called significant genes of <1.
