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Pipeline publication

[…] y database []. The data are publicly available both in the Stanford Microarray Database and in the NIH Gene Expression Omnibus []., Data were filtered to include only spots that met the following criteria for at least 80% of the samples tested: signal intensity 2.5-fold above background in either the Cy5 (sample) or Cy3 (reference) channel, and a regression correlation for the two channels of at least 0.6 across each measured element. A normalization factor was applied so that the mean log2 ratio for each array (sample) was zero, and data for each spot were then median-centered across all observations. Transcripts that differed in abundance in control and infected mice were identified using Significance Analysis of Microarrays []; control and infected samples from each time point were treated as matched pairs. The Fligner-Killeen test for equality of variance [] was used to identify transcripts with more variable expression in the infected mice than the control mice. Selected data from infected samples were normalized to the average of the day-matched control samples, organized using a Self Organizing Map (SOM) algorithm [], and visualized using Java Treeview []. The data files for each array are publicly available at the Gene Expression Omnibus (GEO) database repository (GSE 16803)., The GeneTrail software program [,] was used to identify Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) categories enriched in specified subsets of the data [,]., Differential gene expression identified by microarray analysis was validated by determining the relative quantities of several gene transcripts from infected and uninfected mice using real time RT PCR. Several recent studies have demonstrated that there is no one universal reference gene that has constant, stable expression in all different tissue types [,]. Thus, to identify an appropriate internal control for these experiments, we evaluated the expression of a set of commonly used control genes [], and identifie […]

Pipeline specifications

Software tools SAM, TreeView, GeneTrail
Databases GEO
Organisms Mus musculus, Streptococcus pneumoniae, Mycoplasma pneumoniae