Total RNA was extracted individually from fish skin using QIAGEN RNeasy® Mini Kit following the manufacturer’s recommendations. The total RNA concentration was quantified using a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity and quality checked with the Experion (Automated Electrophoresis Station, Bio-Rad) using the Experion Standard Sens RNA chip (Bio-Rad). Only the samples with an RNA integrity number (RIN) > 8.0 were chosen for further analysis. Transcriptional analysis was carried out using the AquaGenomic Sparus aurata oligonucleotide microarray (SAQ) platform (39). The complete information on this platform and our data is available through the public repository Gene Expression Omnibus (GEO) (accession numbers GPL13442 and GSE162501, respectively) at the United States National Center for Biotechnology Information (NCBI). A transcriptomic analysis was conducted to determine differences at the expression level between control and SDPP groups at the end of feeding trial (95 days). For each experimental group (control; SDPP group) total RNA samples were pooled (n = 3 pools each group; n = 4 fish each pool, n = 1 fish taken at random from each tank for each pool) using the same final concentration (133 ng/µL each pool). One-color microarray was carried out according to the manufacturer’s protocols. Briefly, 200 ng of total RNA was reversed transcribed along with spike-in (Agilent One-Color RNA Spike-In kit, Agilent Technologies, United States). The solution was then used as template for Cyanine-3 (Cy3) labeled cRNA synthesis and amplification with the Quick Amp Labeling kit. cRNA samples were purified using the RNeasy micro kit (Qiagen) according to manufacturer’s instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer. 1.5 mg of Cy3-labeled cRNA with specific activity of >6.0 pmol Cy3/mg cRNA was then fragmented at 60°C for 30 min, and the samples were then mixed with hybridization buffer and hybridized to the array (ID 025603, Agilent Technologies) at 65°C for 17 h, using the Gene expression hybridization kit. Washes were conducted as recommended by the manufacturer, using gene expression wash buffers and a stabilization and drying solution (Agilent Technologies). Microarray slides were scanned with Agilent Technologies Scanner model G2505B. Spot intensities and other quality control features were extracted with Agilent’s Feature Extraction software version 10.4.0.0 (Agilent Technologies). Quality reports were checked for each array. The extracted raw data were imported and analyzed with GeneSpring (version 14.5 GX software, Agilent Technologies). The 75% percentile normalization was used to standardize the arrays for comparisons, and data were filtered by expression. The differential expressed genes (DEGs) were obtained from a gene-level differential expression analysis. Expression values with a p-value < 0.05 were considered statistically significant. The DEGs were grouped according to its fold-change value (p-value < 0.05) and represented using the GraphPad software v7.0 for Windows. The Principal Component Analysis (PCA) was carried out using GeneSpring software (Agilent), four eigenvectors were calculated to describe the aggrupation of the control and SDPP groups in a 3D plot. The gene expression values (log2-expression ratios) were represented by a hierarchical clustering heatmap analysis using MeV software (v4.0), with Pearson distance and average linkage as it was described before (40).

In order to classify the DEGs (both up- and down-regulated) according to its functional annotation, genes were imported into the web-tool Protein ANalysis Through Evolutionary Relationships (PANTHER) classification system (version 13.0) (41). This web resource allows understanding the biological meaning behind large list of DEGs based on their GO classification. For the enrichment analysis, the major over-represented GO were chosen according to a p-value < 0.05 criteria (biological processes; cellular component). The biological interpretation of the DEGs obtained was complemented using the free access databases GeneCards (www.genecards.org) (42) and UniProt (www.uniprot.org) (43).

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