Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

The stability of 12 potential reference genes was examined during larval development in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus) to determine the most suitable genes for qRT-PCR analysis. Transcription levels of genes encoding β-Actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), annexin A2 (ANXA2), glutathione S-transferase (GST), ornithine decarboxylase (ODC), hypoxanthine phosphoribosyltransferase (HPRT1), ubiquitin (UBQ), elongation factor 1 alpha (eEF1A1), 18S ribosomal RNA, and the ribosomal proteins S4 (RPS4) and L13a (RPL13a) were quantitated. Two paralogous genes for ACTB were analyzed in each of both flatfish species. In addition, two paralogous genes for GAPDH were studied in Senegalese sole. RPL13a represented non-orthologous genes between both flatfish species. GeNorm and NormFinder analyses for expression stability revealed RPS4, UBQ and eEF1A1 as the most stable genes in Senegalese sole, Atlantic halibut and in a combined analysis. In all cases, paralogous genes exhibited differences in expression stability.This work suggests RPS4, UBQ, and eEF1A1 genes as useful reference genes for accurate normalization in qRT-PCR studies in Senegalese sole and Atlantic halibut larvae. The congruent results between both species in spite of the drastic differences in larval development suggest that selected housekeeping genes (HKGs) could be useful in other flatfish species. However, the finding of paralogous gene copies differentially expressed during development in some HKGs underscores the necessity to identify orthologous genes.Pleuronectiformes (flatfish) are a broad taxonomic group comprising 11 families and about 500 species worldwide, and some of them are of high commercial interest both in fisheries and aquaculture. Senegalese sole, Solea senegalensis, and Atlantic halibut, Hippoglossus hippoglossus, are two representative species of Pleuronectidae and Soleidae families, respectively. Pleuronectidae (right-eye