G2565c microarray scanner

MiRNA expression profiling was performed using SurePrint™ 8 × 15 K Rat v21 miRNA microarrays (Agilent Technologies, Santa Clara, CA, USA). These microarrays contain 719 mature miRNAs of miRBase v21. All procedures were carried out according to the manufacturer’s recommendations. In brief, a total of 100 ng total RNA from each sample was dephosphorylated by incubation with calf intestinal phosphatase (CIP) at 37 °C for 30 min and denatured with 100% dimethyl sulfoxide (DMSO) at 100 °C for 7 min. Samples were labeled with pCp-Cy3 with the use of T4 ligase at 16 °C incubation for 2 h. Each labeled RNA sample was then hybridized onto an individual subarray, with each array containing probes for 306 miRNAs. Hybridizations were performed in SureHyb chambers (Agilent Technologies) at 55 °C for 20 h with rotation. Arrays were then washed, dried and scanned at a resolution of 3 μm double-pass using an Agilent G2565C Microarray Scanner. Data were acquired using Agilent AGW Feature Extraction software version 10.10.11.

For microarray analysis, a microarray slide for bovine mRNA—Bovine Gene Expression Microarray v2.0, 4 × 44 K (G2519F-#023647, Agilent Technologies), which included 43,713 probes for bovine mRNAs—was used. Hybridized microarray slides were scanned and fluorescence intensities were measured using a G2565C microarray scanner (Agilent Technologies). The obtained data were analyzed using GeneSpring GX software (Agilent Technologies). The data were normalized by 75 percentile shift according to the manufacturer’s instructions and a moderated t-test [31 (link)] with Benjamini–Hochberg multiple testing correction [32 (link)]. The corrected p-value cutoff was set to 0.05.

A custom high-density oligo-microarray (8 × 15 K) from the assembled nucleotide European sea bass sequences was designed and printed using the eArray web tool (Agilent). The array comprised 60-oligomer probes for 14,147 different European sea bass annotated sequences. Total RNA (150 ng) from individuals (n = 8 for each intestine segment: AI, MI, and PI) were labeled with cyanine 3-CTP (Low Input Quick Amp Labeling Kit, Agilent), and 600 ng of each labeled cRNA were hybridized to microarray slides that were analyzed with an Agilent G2565C Microarray Scanner according to the manufacturer's protocol. Data were extracted using the Agilent Feature Extraction Software 11.5.1.1. Data analysis of differentially expressed (DE) genes was carried out with the Genespring GX 13.0 software (Agilent). Functional pathway analysis of DE genes was performed with the Ingenuity Pathway Analysis (IPA) software (http://www.ingenuity.com). For each gene, the Uniprot accession of the annotation equivalent for one of the three higher vertebrates model species in IPA (human, rat, or mouse) was assigned.