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Title: Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues      
Transcriptome or Gene expression
An EST database from immune tissues was used to design the first high density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60mers) were successfully designed for 2716 out of 3482 unique sequences of the database. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida challenged fish at 3 days post-infection. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide and dye bias) factors using one-colour (1C) and two-colour (2C) -labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random effects variance revealed that technical variation was mostly negligible and biological variation represented the main factor, even using pooled samples. One-colour approach performed at least as well as 2C. A relevant proportion of genes turn out to be differentially labelled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defence response were detected at three days post-challenging. Overall design: An unbalanced hierarchical design was planned to evaluate the sources of variation in microarray analysis. This design enabled us to consider most sources of noise in microarray analysis and evaluate their weight in the variability of the signal using an equilibrate number of replicates at each level and a not too large number of microarrays. According to this hierarchy, the following within slide sources of variation were assessed from top to bottom: biological (B), RNA extraction (E), labeling (L) and hybridization (H). Additionally, we studied variation between slides (S), the bias produced by labeling with different dyes (Cy3 and Cy5) and compared the performances of one-colour (1C) and two-colour (2C) -labeling approaches. A total of 24 microarrays grouped in three 8x15k slides were used for this design. One pool of five individuals was used as control and other two other pools of five individuals each (B1 and B2) were used as biological replicates. Three replicates were used for evaluating the variation associated with RNA extraction (E1, E2, E3), labeling (L1, L2, L3) and hybridization (H1, H2, H3) within slide. Some of these replicates were in turn hybridized in different slides to assess interslide variation. Three 2C hybridizations with dye swapping (6 microarrays) were performed in the same slide for comparison of 1C and 2C approaches and to analyze dye bias. Normalization within each microarray was carried out using the loess method. Normalization using all microarray data was done by performing the Aquantile method implemented in the limma package. To estimate the variation associated with replicates at the different steps of microarray analysis or to different slides we applied the Pearson correlation coefficient using log2 absolute treatment and treatment/control ratio values. Also an ANOVA of random effects was used to split the total variance into its components. For Dye bias evaluation and 1C vs. 2C comparisons we f estimated the proportion of common differentially expressed (DE) genes after Bonferroni correction at P 1.5, 2 and 3. The degree of concordance between the different dyes and between 1C and 2C approaches was estimated as the proportion of common DE genes at each P and FC.
Scophthalmus maximus
National Center for Biotechnology Information
NCBI BioProject