tissue: Skin age: juveniles, 155 g treatment: control
Treatment protocol
Gilthead sea bream (Sparus aurata) were obtained from an aquaculture company (Valencia, Spain) and transported to the on-grown facilities of IRTA in Sant Carles de La Ràpita (Tarragona, Spain). All animal experimental procedures were conducted in compliance with the experimental research protocol approved by the Committee of Ethics and Animal Experimentation of the Institut de Recerca i Tecnologia Agroalimentàries (IRTA) and in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. After transport, fish received three treatments with hydrogen peroxide and two treatments with formaldehyde (150 ppm, during one hour each) to treat injuries and avoid the presence of any parasite. After the on-growing period of 105 days, 150 fish (BW = 40.26 ± 0.14 g) were randomly distributed in six aerated 450 L square-shaped tanks with smoothed corners, using an IRTAmar® recirculation system (5-10 % water replacement per day) under natural photoperiod. Water temperature (21-28 °C), oxygen (6.8 ± 1.7 mg/L) and pH (7.5 ± 0.01) were daily controlled, whereas salinity (35‰) as well as ammonia (0.13 ± 0.1 mg NH4+ L−1) and nitrite (0.18 ± 0.1 mg NO2- L−1) levels were weekly monitored.The nutritional assay was carried out in triplicate tanks with an initial density of 2 kg/m3 (25 fish per tank). Fish were fed both diets two times per day at the daily rate of 3.0% of the stocked biomass, which approached apparent satiation. At day 65 of feeding, four fish were randomly selected from each tank, euthanized with anaesthetic (MS-222, Sigma-Aldrich) and a section from the skin was sampled and stored in RNAlater™, incubated overnight at 4 °C, and stored at -80 °C until analysed.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from skin using the RNeasy® Mini Kit (Qiagen, Germany). Total RNA was eluted in a final volume of 35 μL nuclease-free water and treated with DNAse using the DNA-freeᵀᴹ DNA Removal Kit according to manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, Lithuania). Total RNA concentration and purity were quantified using a Nanodrop-2000® spectrophotometer (Thermo Scientific, USA) and stored at -80 °C until analysis. Prior to hybridization with microarrays, samples were diluted to 133.33 ng/µL concentration and checked for integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Spain). All the RNA analysed in this study were selected by the criteria of a RIN value > 8.5.
Label
Cy3
Label protocol
One-color microarray was carried out according to 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, USA). Then, the solution was used as template for Cyanine-3 (Cy3) labeled cRNA synthesis and amplification with the Low Input Quick Amp Labelling kit (Agilent). Samples of cRNA were purified using the RNeasy micro kit (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
Hybridization protocol
The hybridization protocol was carried out according to Agilent's instructions. Briefly, 1.65 μg of Cy3-labeled cRNA (specific activity >6.0 pmol Cy3/mg cRNA) was fragmented at 60°C for 30 min in a total reaction volume of 55 µl containing 25X Agilent fragmentation buffer and 10X Agilent blocking agent. On completion of the fragmentation reaction, 55 µl of 2X GEx hybridization buffer was added to the fragmentation mixture and hybridized to S. aurata array (ID 024502, Agilent) at 65°C and 10 rpm for 17 hours in a rotating hybridization oven.
Scan protocol
After hybridization, microarrays were washed 1 min at room temperature (RT) with GEWash Buffer 1 (Agilent), 1 min with GE Wash buffer 2 (Agilent) pre-incubated at 37°C, 45 s at RT with Acetonitrile (Sigma, St Louis, MO, USA), and 30 s at RT with stabilization and drying solution (Agilent). Slides were scanned immediately afterwashing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area: 61 x21.6 mm; Scan resolution: 5 µm; Dye channel set to green; Green PMT: 100%). Spot intensities and other quality control features were extracted with Feature Extraction software version 10.4.0.0 (Agilent).
Description
Gene expression for control group pool 3
Data processing
For microarray analysis, a percentile shift normalization was carried out and then data were filtered by expression (standard deviation) among groups. Then, gene-level analysis was conducted in order to obtain the median of signal intensities from spots within a probe set. Statistical test (t-test unpaired) was done with GeneSpring software GX 14.3 to obtain the differentially expressed genes (p-value < 0.05) between control and treatments.
Submission date
Dec 02, 2020
Last update date
Mar 31, 2021
Contact name
Felipe E. Reyes-López
Organization name
Universitat Autonoma de Barcelona
Department
Cell Biology, Physiology and Immunology
Lab
AQUAB-FISH
Street address
Animal Physiology Unit, Department of Cell Biology, Physiology and Immunology, Faculty of Biosciences, Universitat Autònoma de Barcelona
