Comment[ArrayExpressAccession]	E-GEOD-50964
MAGE-TAB Version	1.1				
Public Release Date	2013-09-25
Investigation Title	Differential expression of Dictyostelium discoideum AX2 upon infection with wt L. pneumophila JR32 vs. uninfected 24h p.i. (timecourse experiment)				
Comment[Submitted Name]	Differential expression of Dictyostelium discoideum AX2 upon infection with wt L. pneumophila JR32 vs. uninfected 24h p.i. (timecourse experiment)
Experiment Description	Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila in comparison to uninfected cells was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up- or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strain used in this study was L. Pneumophila Philadelphia I JR32. The strain was grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 cme2 cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. After different time intervals of incubation (1, 3, 6, 24, and 48 h) the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells. Usually two or three parallel cultures for the experiment and the control were inoculated per infection. The percentage of infected cells was determined by in situ hybridization with Legionella-specific 16S rRNA probes. The average from three independent determinations was 34, 42, 42, 57 and 82% of infected Dictyostelium cells after 1, 3, 6, 24 and 48 h, respectively.				
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl			
Person Last Name	Eichinger	Farbrother	Konertz	Eichinger	
Person First Name	Ludwig	Patrick	Roman	Ludwig	
Person Email	ludwig.eichinger@uni-koeln.de				
Person Affiliation	Institute for Biochemistry 1				
Person Phone	+49 221 478 6928				
Person Address	Institute for Biochemistry 1, Joseph-Stelzmann-Strasse 52, Cologne, Germany				
Person Roles	submitter				
Protocol Name	P-GSE50964-1	P-GSE50964-3	P-GSE50964-4	P-GSE50964-2	P-GSE50964-5
Protocol Description	For each individual comparison, 19 microarrays were hybridised and analysed. Two image pairs were produced per microarray slide, one with high laser intensity so that signals for most probes and also some saturated signals were obtained and a second one with lower laser intensity so that none of the signals was saturated. This way the dynamic range of the measurement was expanded. To handle the import and export of microarray data to different analysis programs an Excel Add-In, Array tools, was programmed in Visual Basic. Upon import of two data files of the same microarray scanned with different laser powers, the saturated spots of the high laser power scan were replaced by non-saturated spots from the low laser power scan. In addition, the import also performed data filtering by flagging SpotReport controls, negative controls, empty spots, spots where only spotting solution was printed and spots whose intensities were below or equal to zero as "Bad". Fluorescence ratios were normalised by LOWESS-normalisation using R1.6.2 (BioConductor, http://www.bioconductor.org/). The normalized M values (M = log2 (Intensityexperiment/Intensitycontrol) were transferred into a new worksheet for significance analysis of microarrays (SAM) analysis (Tusher et al., 2001). At the transfer all probes spotted in higher replicates were reduced to double spots by averaging. Furthermore, all probes that were flagged "Found" in less than half of the spots, were excluded from SAM analysis. Differentially regulated genes were identified using the one-class SAM method calculating 1000 permutations. The SAM program not only identifies the differentially regulated genes, but also predicts the number of false positives. These are the genes that are falsely reported as differentially expressed. This feature was used in all microarray experiments to set the significance level such, that the 90th percentile of the false discovery rate (FDR) was minimal. See also Farbrother et al., Cell. Microbiol., 3:438-456, 2006. ID_REF =  AR =  AC =  SR =  SC =  Name =  GID =  Diameter =  Flags =  VALUE = Normalised M-value; M=log2(Intensity_Exp/Intensity_Con) A norm = Normalised A-value; A=log2 times square root of (I_Exp x I_Con) M raw =  A raw =  Exp Norm = Normalised intensity value of experiment Con Norm = Normalised intensity value of control Con Median = Median intensity value of control Con SD =  Con B Median = Median background intensity value of control Con B SD =  Con % > B + 2 SD =  Con F % Sat. =  Con SignalNoiseRatio =  Exp Median = Median intensity value of experiment Exp SD =  Exp B Median = Median background intensity value of experiment Exp B SD =  Exp % > B + 2 SD =  Exp F % Sat. =  Exp SignalNoiseRatio =  Exp Log Ratio =  Con N Median =  Exp N Median =  Exp N Log Ratio = 	Fairplay indirect labelling kit.	Cy3 and Cy5 labelled targets were mixed, ethanol precipitated and dissolved in 65 M-5l of hybridisation buffer (Noegel et al., 1985) with 500 M-5g/ml Fish sperm DNA (Roche, Mannheim, Germany) and 2 M-5M Oligo dA 18-mer. The hybridisation mix was heated to 80 M-0C for 10 min, applied to the microarray under a cover-slip and incubated in a hybridisation chamber (Corning, New York, USA) for 15 hours at 37M-0C. Post-hybridisation washes were performed twice with 2x SSC, 0.1% SDS and once with 0.1x SSC, 0.1% SDS for 5 min each, five times with 0.1x SSC and once with with 0.01x SSC for 5 sec each and dried by centrifugation at 235xg for 5 min. See also Farbrother et al., Cell. Microbiol., 3:438-456, 2006.	RNA was isolated using the protocol for the isolation of cytoplasmic RNA of the RNeasy mini kit (Qiagen AG).	Signal detection was performed with the ScanArray 4000XL confocal laser scanner (PerkinElmer Life Sciences, Wellesley, USA). Images for Cy3 and Cy5 were obtained, spots were detected and quantified with ScanArray Express v2.2 (PerkinElmer Life Sciences), then manually inspected and if necessary corrected. See also Farbrother et al., Cell. Microbiol., 3:438-456, 2006.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	TREATMENT				
Experimental Factor Type	treatment				
Publication Title	Dictyostelium transcriptional host cell response upon infection with Legionella.				
Publication Author List	Farbrother P, Wagner C, Na J, Tunggal B, Morio T, Urushihara H, Tanaka Y, Schleicher M, Steinert M, Eichinger L				
PubMed ID	16469056				
Publication DOI	10.1111/j.1462-5822.2005.00633.x				
Comment[SecondaryAccession]	GSE50964				
Comment[GEOReleaseDate]	2013-09-25				
Comment[ArrayExpressSubmissionDate]	2013-09-18				
Comment[GEOLastUpdateDate]	2013-09-25				
Comment[AEExperimentType]	transcription profiling by array				
SDRF File	E-GEOD-50964.sdrf.txt