tank: 14 protocol: Low ARA, high VA time: 17 dph tissue: Whole larvae
Treatment protocol
Experimental diets consisted of four different enriched rotifer cultures. Two oil mixtures were made, named Control and High ARA. Control contained 90% EPAX4510 and 10% EPAX5010, while High ARA contained 65% Cargill ARA oil, 5% EPAX4510 and 30%EPAX5010. The mixtures were then divided in two fractions. VA in the form of retinylpalmitate (Sigma-Aldrich, Oslo, Norway) was added to one fraction from each oil mixture (5 g retinylpalmitate L-1 oil). This gave four different mixtures Control, Control+VA, High ARA and High ARA+VA. Four enriceh The rotifer cultures (5 to 7-days batch cultures) were kept in 2 m3 conical tanks at 24°C with rotifer densities ranging between 800 and 2000 rotifers•ml-1, and fed four times•hour-1 on a daily basis. The diet consisted of dry baker’s yeast (0.11-0.18 g•million rotifers-1) and Rotifer Diet® (Reed Mariculture Inc., CA, USA, 0.3-1.5 g•million rotifers-1). Approximately 1.2 mill rotifers were fed to each tank at the start of the feeding trial and this was increased according to Austevoll standard feeding protocol (van der Meeren T, Mangor-Jensen A, Pickova J. 2007 Aquaculture. 265: 206-217)
Growth protocol
The rotifer cultures (5 to 7-days batch cultures) were kept in 2 m3 conical tanks at 24°C with rotifer densities ranging between 800 and 2000 rotifers•ml-1, and fed four times•hour-1 on a daily basis. The diet consisted of dry baker’s yeast (0.11-0.18 g•million rotifers-1) and Rotifer Diet® (Reed Mariculture Inc., CA, USA, 0.3-1.5 g•million rotifers-1). The Atlantic cod larvae were kept in 16 50L cylindrical, flat bottomed tanks with center tubes covered with 250 µm filter, at the Institute of Marine Research, Austevoll Research Station (Storebø, Norway). Water of the same quality and salinity as above was used, with an initial temperature of 9.4 °C. Temperature was increased to 12 °C at 13 dph over a three day period to avoid temperature stress. Modest oxygen bubbling around the center tube was used to ensure even distribution of live prey in the tanks. A steady flow of daily fresh made algal “soup” was added to create green water and optimal turbidity (1 formazine turbidity units) in the tanks. Larvae were hatched according to protocols used at Austevoll marine station (van der Meeren T and Ivannikov VP. 2006, Aquac Res. 37: 898-913).
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from pooled larvae samples using BioRobot® EZ1 and RNA Tissue Mini Kit (Qiagen). DNAse treatment step was implemented according to the protocol (Qiagen). Total RNA quantity and quality were determined using the NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), while RNA integrity (RIN) was analyzed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip® kit (Agilent Technologies, Palo Alto, CA, USA). All samples had 260/230 and 260/280 ratios of > 2.0 and RIN of > 8, respectively
Label
Cy3
Label protocol
Labeling was performed by Roche NimbleGen (Roche NimbleGen , Madison, WI), following their standard operating protocol (Nimblegen Arrays User’s Guide: Gene Expression Arrays v5.0, 2010 Roche Nimblegen)
Hybridization protocol
Hybridization was performed by Roche NimbleGen (Roche NimbleGen , Madison, WI), following their standard operating protocol (Nimblegen Arrays User’s Guide: Gene Expression Arrays v5.0, 2010
Scan protocol
Scanning was performed by Roche NimbleGen (Roche NimbleGen , Madison, WI), following their standard operating protocol (Nimblegen Arrays User’s Guide: Gene Expression Arrays v5.0, 2010
Description
Sample name: 2010-369/112 Whole animal
Data processing
signal intensities extraction and global quantile normalization were done according to Nimblegen Arrays User’s Guide: Gene Expression Arrays v5.0, 2010 Roche Nimblegen. The analysis of the Nimblegen microarray data was performed in the R environment for statistical computing and graphics (R Development Core Team, 2009; version 3.0.2) as implemented in RStudio (Integrated development environment for R, 2013; version 0.98.507) using software from the Bioconductor project [42]. Following an approach previously described, the pdInfoBuilder package protocol version 1.26.0 (http://www.bioconductor.org/packages/release/bioc/html/pdInfoBuilder.html) was used to create an annotation package for the Nimblegen custom design file “100610_GM_FY_EXP.ndf”. Raw expression values of the XYS files were read into R using the oligo package protocol version 1.26.6. (http://bioconductor.org/packages/release/bioc/vignettes/oligo/inst/doc/V2NExpression.pdf) Prior to statistical analysis, the microarray data was further processed applying RMA background subtraction and normalization. (gene calls) were estimated from triplicate probes using multichip average (RMA) as previously described (Irizarry et al., 2003. Nucleic Acids Res. 31 and Irizarry et al., 2003 Biostatistics, 4: 249-264) Gene calls with large variation (SE > 0.8) between triplicate probes were omitted from the dataset.
Vitamin A and arachidonic acid altered the skeletal mineralization in Atlantic cod larvae (Gadus morhua) without any interactions on the transcriptional level
