For RT qPCR, 1 ug of column purified total RNA per sample was reverse transcribed into cDNA using the VersoTM cDNA kit, following manufacturers instructions, using a mixture of random hexamers and anchored oligo dT at 3,1. Negative controls were performed to check for genomic DNA contamination. A similar amount of cDNA was pooled from all samples and the remaining cDNA was then diluted 20 fold with water. RT qPCR analysis used rela tive quantification with the amplification efficiency Inhibitors,Modulators,Libraries of the primer pairs being assessed by serial dilutions of the cDNA pool. qPCR amplifications were carried out in duplicate in a final volume of 20 uL containing either 5 uL or 2 uL diluted cDNA, 0. 5 uM of each primer and 10 uL AbsoluteTM QPCR SYBR Green mix. Amplifications were carried out with a systematic negative control.

The qPCR profiles contained an initial activa tion step at 95 C for 15 min, followed Inhibitors,Modulators,Libraries by 30 to 40 cycles, 15 s at 95 C, 15 s at the specific primer pair annealing temperature and 15 s at 72 C. After the amplification phase, a melt curve of 0. 5 C increments from 75 C to 90 C was performed, enabling confirmation Carfilzomib of the amplification of a single product in each reaction. RT qPCR product sizes were checked by agarose gel electrophoresis and the identity of amplicons of newly designed primers was confirmed by sequencing. Lipid extraction and fatty acid analyses Total lipids from six fish per treatment were extracted and determined gravimetrically from 1 2 g of liver by Ultra Turrax homogenisation in 20 volumes of chloroform methanol.

Fatty acid methyl esters were prepared by acid catalysed transesterification of total lipids. Following purification, FAME were separated and quantified by gas liquid chromatography using a Thermo Fisher Trace GC 2000 equipped with a fused silica capillary column with hydrogen as carrier gas and using on column injection. The temperature gradient Inhibitors,Modulators,Libraries was from 50 to 150 C at 40 C min and then to 195 C at 1. 5 C min and finally to 220 C at 2 C min. Individual methyl esters were identified by comparison with known standards. Data were collected and processed using the Chromcard for Windows computer package. Statistical analysis Microarray hybridisation data were analysed in Gene Spring GX version 10. 0. 2 by two way ANOVA, which examined the explanatory power of the variables diet and genotype, followed by Gene Ontology enrichment Inhibitors,Modulators,Libraries analysis, at a significance level of 0.

05. No multiple test correction was employed as previous ana lyses, confirmed by RT qPCR, indicate that such correc tions are over conservative for this type of data. Gene expression results assessed by RT qPCR were analysed by the Ct method using the relative expression software tool, employing a pair wise fixed reallocation randomisation test with efficiency correction, to determine the statistical significance of expression ratios between two treatments.