Design and Performance of a Turbot (Scophthalmus maximus) Oligo-microarray Based on ESTs from Immune Tissues

An expressed sequence tag 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 (60 mers) were successfully designed for 2,716 out of 3,482 unique sequences of the database. An Agilent custom oligo-microarray 8 × 15 k (five replicates/gene; eight microarrays/slide) was constructed. 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 postinfection. Only 48 out of 2,716 probes did not show signal of hybridization on the 32 microarrays employed, thus demonstrating the consistency of the bioinformatic applications of our database. 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 1C and 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-color approach performed at least as well as 2C, suggesting their usefulness due to its higher design flexibility and lower cost. A relevant proportion of genes turn out to be differentially labeled 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/defense response were detected at 3 days postchallenging.