Computational protocol: Salmonella induces prominent gene expression in the rat colon

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Protocol publication

[…] Signal intensities for each spot were quantified using Feature Extraction 8.1 (Agilent Technologies). The data of the time course infections study are available in Additional File and have been deposited in NCBIs Gene Expression Omnibus [] and are accessible through GEO Series accession number GSE7496. Median density values and background values of each spot were extracted for both the experimental samples (Cy5) and the reference samples (Cy3). Quality check was performed for each microarray using both LimmaGUI package in R from Bioconductor [] and Microsoft Excel. Data was exported into GeneMaths XT (Applied Maths, Sint-Martens-Latem, Belgium) for analysis. We discarded spots with an average intensity, over all arrays, of Cy5 lower than 2-fold above average background. Then, the Cy5 intensities were normalized against the Cy3 reference as described before []. The gene expressions of duplicate arrays were averaged. Array data of non-infected rats, killed on section day 1 and 6 were highly comparable and could therefore be considered as one group and were averaged. For unknown reason, arrays of non-infected rats killed on day 3 showed reduced expression of 14 mast cell protease genes when compared with non-infected rats of both days 1 and 6, which were highly comparable. Therefore, we decided not to include the non-infected rats of day 3. Cluster analysis and Principle component analysis were performed using GeneMaths XT. Infected/control ratio's between 0–1 were expressed as the negative inverse (-1/value) for easier interpretation. Genes that changed more than 2-fold in comparison with controls at one of the time points studied were selected for pathway analysis. Pathway analysis was performed using two pathway programs, MetaCore (GeneGo Inc, St. Joseph, MI)[]and ErmineJ [], using Agilent gene annotation (Agilent Technologies, version 20060331). Processes were identified using statistical over-representation in both pathway programs. Since only 40% of the genes were annotated to GO processes in both pathway programs, processes with a p-value < 0.001 were manually supplemented with non-annotated genes with FC > 2 using biological databases (BIOcarta, SOURCE, GenMAPP, KEGG) and scientific literature. [...] Real-time Quantitative RT-PCR (Q-PCR) was performed on individual samples (n = 8 per group). 1 μg of RNA of all individual samples was used for the cDNA synthesis using the iScript cDNA synthesis kit of Bio-Rad Laboratories (Veenendaal, The Netherlands). Real-time reactions were performed by means of the iQ SYBR Green Supermix of Bio-Rad using the MyIQ single-color real-time PCR detection system (Bio-Rad). Each reaction (25 μl) contained 12.5 μl iQ SYBR green supermix, 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 8.5 μl RNase-free water and 2 μl diluted cDNA. The following cycles were performed 1× 3 min at 95°C, 40 amplification cycles (40× 10 s 95°C, 45 s 60°C), 1× 1 min 95°C, 1× 1 min 62°C and a melting curve (80× 10 s 55°C with an increase of 0.5°C per 10 s). A negative control without cDNA template was run with every assay. The optimal melting point of dsDNA (Tm) and the efficiency of the reaction were optimized beforehand. Data were normalized against the reference genes Ribosomal protein S29 (Rps29), ADP-Ribosylation Factor 1 (Arf1) and β-actin. Rps29 and Arf1 were chosen on the basis of microarray data which showed similar expression levels for all microarrays, β-actin was chosen as this is a well accepted reference gene. Primers were designed using Beacon designer 4 (Premier Biosoft International, Palo Alto, CA). For sequences see Additional File . A standard curve for all genes including reference genes was generated using serial dilutions of a pooled sample (cDNA from all reactions). mRNA levels were determined from the appropriate standard curve. Samples with mRNA levels below the lowest standard value, and thus below detection level, were given half the value of this lowest standard. Analysis of all individual samples was performed in duplicate. […]

Pipeline specifications

Software tools limmaGUI, MetaCore, ErmineJ, GenMAPP, Beacon Designer
Databases GEO KEGG BioCarta
Applications Gene expression microarray analysis, qPCR
Organisms Rattus norvegicus, Salmonella enterica subsp. enterica serovar Enteritidis
Diseases Colonic Neoplasms, Infection, Pancreatic Neoplasms, Salmonella Infections
Chemicals Calcium, Glutathione