Normalization strategies for gene expression studies by real-time PCR in a marine fish species, Scophthalmus maximus

• Published in: Marine Genomics
• Main Authors: Urbatzka, R., Galante-Oliveira, S., Rocha, E., Castro, L. F. C., Cunha, I.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Elsevier 2013
• Subjects: Normalization strategies; Peroxisome proliferator-activated receptor agonist; Real-time quantitative RT-PCR; Reference genes; Scophthalmus maximus; Teleost fish
• Online Access: http://hdl.handle.net/10773/24289; https://doi.org/10.1016/j.margen.2013.02.001

Thorough evaluation of normalization approaches is a fundamental aspect in real-time quantitative RT-PCR experiments to avoid artificial introduced intergroup variations. In our study, we tested three normalization strategies in an experimental data set derived from a toxicological exposure of Scophthalmus maximus to the peroxisome proliferator-activated receptor alpha (PPARα) agonist WY-14643. Juvenile turbots were exposed by repeated injections to 5. mg or 50. mg WY-14643/kg, and liver samples were taken at day 1, 7 and 21. Specifically, the mRNA expression of peroxiredoxin 5 (prdx5) was normalized to the cDNA content, to the mRNA expression of single reference genes (b-actin, b-act; elongation factor 1 α, ef1a; glyceraldehyde-3-phosphate dehydrogenase, gapdh; ribosomal protein L8, rpl8; tata-box binding protein, tbp; tubulin beta 2C chain, tubb2c; ubiquitin-conjugating enzyme E2L 3, ub2l3) or to a combination of multiple reference genes using geNorm, BestKeeper or NormFinder algorithms.Four single reference genes ( ef1a, rpl8, tubb2c, tbp) did not show any significant differences between the treatment groups over time, while significant intergroup variations were observed for cDNA content, gapdh, b-act and ub2l3. The normalization of prdx5 to the valid (not altered) single reference genes led to significant up-regulated ( prdx5/. rpl8), not-regulated ( prdx5/. ef1a; prdx5/. tbp) or down-regulated ( prdx5/. tubb2c) mRNA expression pattern. The multiple reference gene approaches resulted in different rankings and combinations of the most stable expressed reference genes (geNorm: ef1a>. rpl8>. b-act; BestKeeper: ub2l3>. gapdh>. ef1a; NormFinder: b-act>. ef1a). However, the normalization with the three multiple reference gene procedures demonstrated consistent expression pattern with a significant up-regulation of prdx5 in response to the higher concentration after 21. days.Concluding, even if not yet established as "gold" standard for expression profiling in environmental toxicology or physiology ...