Gene expression analysis by quantitative real-time PCR

The gene expression analysis was studied in three selected dietary groups, including low (diet 1), mid (diet 1·6) and high (diet 3·5) EPA + DHA levels. Candidate genes involved in trace mineral metabolism (HAMP; TFR, transferrin receptor; Met-B, metallothionein-B), stress response (CAT, catalase; SOD, superoxide dismutase; Gpx1, glutathione peroxidase 1; Gpx4b, glutathione peroxidase 4b; Gpx7, glutathione peroxidase 7; GR, glutathione reductase; GST1, glutathione S-transferase 1; FAS, fatty acid synthase; G6PD, glucose-6-phosphate dehydrogenase; SePP, selenoprotein P and HSP70, heat shock protein 70) and inflammatory markers (IFN-γ, interferon gamma; TNF1α, tumour necrosis factor 1 alpha; TGF-β 1, transforming growth factor β 1 and IL4/13a, interleukin 4/13a) were analysed in the liver. The procedure for RNA extraction, reverse transcription and quantitative PCR (qPCR) followed were as described in Hundal et al. ⁽³⁴⁾. In brief, the total RNA was extracted from liver tissue using EZ1 RNA Universal Tissue Kit (Qiagen) and the BioRobot EZ1 according to the manufacturer’s descriptions. Quality and integrity of RNA were assessed with the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). A two-step real-time PCR protocol was followed to assess the mRNA transcriptional levels of the selected target genes. The stability of the reference genes (geometric mean of both βact and elf1α) and mean normalised expression of the target genes were calculated using CFX Maestro software (Bio-Rad CFX maestro version 1.1, Bio-Rad laboratories). The details of the qPCR primers used for amplification of the reference and target genes are provided in Table 3.

Primers used for quantitative PCR

CAT, catalase; SOD, superoxide dismutase; Gpx1, glutathione peroxidase 1; Gpx4b, glutathione peroxidase 4b; Gpx7, glutathione peroxidase 7; GR, glutathione reductase; GST, glutathione S-transferase; SePP, selenoprotein P; Met-B, metallothionein-B; HAMP, hepcidin antimicrobial peptide; TFR, transferrin receptor; FAS, fatty acid synthase; G6PD, glucose 6 phosphate-1 dehydrogenase; HSP 70, heat shock protein 70; IFN-γ, interferon gamma; TGF-β 1, transforming growth factor beta 1; IL4/13a, interleukin4/13a; EF1αb, elongation factor alpha b.