Technical replicates to determine array-to-array reproducibility.

Technical replicates, consisting of four independent hybridizations of the same labelled cRNA, were performed to determine array-to-array reproducibility. After normalization, correlation index between all replicates exceeded 98%. In this study, we analyzed reproducibility of Agilent-016251 Sparus aurata Oligo Microarray based on single-colour detection (Cyanine-3 only). A RNA sample, prepared mixing four different larval stages of gilthead sea bream, was labelled in according to Agilent One-Color Microarray-Based Gene Expression Analysis protocol. The same labelled cRNA was used for four independent fragmentation reactions and then hybridized spanning all array positions of 4x44K platform. Microarrays are scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides are scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software creates a unique ID for each pair of XDR scans and saves it to both scan image files. Feature Extraction 9.5 uses XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended Background-Subtracted Signal.