Computational protocol: Xylella fastidiosa gene expression analysis by DNA microarrays

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Protocol publication

[…] The slides were submitted to fluorescence reading in a model GMS 418 Arrayer Scanner (Affymetrix Inc.) under different wavelengths - 550 nm (Cy3) and 650 nm (Cy5). The location and identity of each gene on the slide was defined in a text file, created with the aid of the CloneTracker2 program (BioDiscovery). The signal was quantified through ImaGene software (v. 4.1, BioDiscovery), in which two images from the Cy3 and Cy5 fluorescent dyes were overlapped and the spots classified according to morphology and intensity. The computer displays an electronic symbol as a false-color image where a red or green spot corresponds to expression of a gene in sample 1 or 2, respectively, while a yellow-orange spot indicates that the gene was expressed at similar levels in both samples. For transformation of data, the background signal was discounted from the signal of each spot using the local background obtained by the GeneSight (BioDiscovery) program. The transformation sequence included background correction, omitted flagged spots, combined replicates and floor, by adding a shifted Log transformation and ratio.Data obtained from the intensity ratio of the signal measured by the Cy5 (experiment) over the Cy3 (control) were normalized according to the average intensity of the total signal. We used all the genes in our dataset to calculate normalization since this assumes that the majority of the measured genes were not differentially regulated. The normalization procedure is a suitable approach for minimizing variations so that a common base for comparison is established. There are a number of reasons that justify data normalization, these including unequal quantities of starting RNA, differences in labeling or detection efficiency among the different fluorescent dyes used, and systematic bias in measured expression levels (). However, current normalization methods are not applicable to all conditions. Normalization can be carried out in several different ways, such as within the slide in order to adjust dye incorporation efficiency, between two slides for dye swap experiments and across slides for repetition of the same experiments (). In the latter case, application would be to the entire data set (overall normalization), instead of to a particular physical data subset or sub grid (local normalization).Final intensity of hybridization was determined in all the experiments from six replicates per data point, and is representative for three independent determinations (slides) from each media culture. Replicates in duplicate within each slide were combined by the median of their values, whereupon statistical analysis was carried out using the SAM method (Significance Analysis of Microarrays). This method is based on t-test statistics and is employed to calculate the false discovery rate (FDR) and gene error chance (q-value) (). Significant variations in expression of those genes related to X. fastidiosa metabolism were compared when cultivated in liquid modified BCYE and liquid XDM2 median. […]

Pipeline specifications

Software tools ImaGene, SAM
Application Gene expression microarray analysis
Organisms Xylella fastidiosa