Computational protocol: Transcriptome response analysis of Arabidopsis thaliana to leafminer (Liriomyza huidobrensis)

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Protocol publication

[…] Affymetrix microarrays (A. thaliana ATH1 genome arrays) containing 22,810 probe sets were used in our experiments. Labeling and hybridization of the ATH1 microarrays (one sample per chip) was performed according to the manufacturer’s instructions (http://www.affymetrix.com/support/technical/manuals.affx). Expression Analysis Technical Manual For HT Array Plates Using the GeneChip® Array Station. Quality control was carried out according to Affymetrix microarray standards, which indicated that all samples met required criteria with respect to control signals, house-keeping gene signals, “present” percent numbers, and low background and noise values. The probe arrays were scanned and further analyzed using GENESPRING software (version 5.0; Silicon Genetics). Normalization per gene and per chip of the log2 values was performed to allow comparison of the three independent replicates performed for each set of experiments. In addition, normalization was performed separately for each experiment and plant tissue for all measurements using the flags “present”, “marginal”, or “absent” assigned by Affymetrix treatment of the arrays. However, only those transcripts that were declared “present” or “marginal” in at least two of three chips were taken into account. To identify differentially-expressed genes in each treatment, SAM analysis (Significance Analysis of Microarrays software package) was conducted on A. thaliana triplicate samples between treatments and controls using a q-value ≤ 0.05 and fold change ≥ 2 as cut-off criteria. We searched for GO information for the differently-expressed probe sets using EasyGO software (http://bioinformatics.cau.edu.cn/easygo/category_treeBrowse.html). For biological process searching, we applied χ2 tests with a false discovery rate (FDR)-adjusted cutoff of P < 0.0001. Cluster 3.0/Treeview software [] was used to group and display genes with similar expression profiles (http://rana.Stanford.EDU/software/). Hierarchical clustering using default options and the uncentered correlation similarity metric was performed on the normalized data. The microarray data in MIAME-compliant format have been deposited in the GEO database (GEO record number GSE38281). […]

Pipeline specifications

Software tools GeneSpring GX, EasyGO, TreeViewX
Application Phylogenetics
Organisms Arabidopsis thaliana, Liriomyza huidobrensis