Dataset features


Application: Gene expression microarray analysis
Number of samples: 67
Release date: May 15 2007
Last update date: Mar 16 2012
Access: Public
Diseases: Chlamydia Infections, Infection, Chlamydophila Infections
Chemicals: Iron
Computational protocol: Primer3, geNorm, MedGen
Dataset link Gene expression profiles of Chlamydophila pneumoniae during the acute and iron mediated persistent infection

Experimental Protocol

A 50mer oligo array containing all open reading frames of the genome projects of CWL029 (, AR39 ( and J138 ( was designed. To ensure maximum specificity, oligo sequences were compared for homology against the human genome using the BLAST algorithm. Design and spotting was done by MWG-Biotech AG (Germany). Cross-hybridization of eukaryotic RNA was confirmed to be marginal by labeling and hybridizing eukaryotic total RNA with the described procedure. Arrays were blocked prior to hybridization by incubation for 45 min at 42°C in blocking solution (4x SSC, 0.5% SDS, 1% BSA), washed five times with H20 and dried by centrifugation (1000 rpm, RT, 2 min). Microarray experiments were done as two-color hybridizations. In order to compensate for dye-specific effects and to ensure statistically relevant data, a color swap was performed. Reverse transcribed cDNA pools were mixed with hybridization buffer (Ambion), denatured, snap-cooled, pipetted onto the array using Lifter Slips (Erie, USA) and placed in a sealed humidified hybridization chamber (Scienion, Germany) at 42° C for 48 hours without shaking. After hybridization the array was washed and dried according to the Ambion protocol. Scanning of microarrays was performed with 5 μm resolution using a microarray laser scanner (Agilent). Features were extracted using the Agilent Technologies image analysis software (Version A7.5) using default settings for non-Agilent microarrays. A local background subtraction method was used and the background was adjusted globally to zero. Dye normalization was done using the rank consistency method and applying a local weighted regression normalization (LOWESS). The ratio between both channels, the log ratio error and the p-values were calculated using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Buildt 4.2. Transcriptome analysis was carried out with two biological replicates for 36 hpi and at least three biological replicates for all other time points. Samples were derived from independent infections, RNA preparations, labeling reactions and hybridizations. We selected genes using a 1.8-fold expression cut-off. All data of the individual sets comprising different time points were combined as Ratio Experiments using negative polarity for the color swap dye-reversal hybridizations. This was achieved by combining the Ratio Profiles, representing single hybridizations, and using an error weighted average based on p-values.








André Mäurer

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