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

Carlos Infante^*¹ , Makoto P Matsuoka^*² , Esther Asensio¹ , José Pedro Cañavate¹ , Michael Reith² and Manuel Manchado¹

¹IFAPA Centro El Toruño, CICE, Junta de Andalucía, Camino Tiro de pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain

²Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada

author email corresponding author email* Contributed equally

BMC Molecular Biology 2008, 9:28doi:10.1186/1471-2199-9-28


Published:	6 March 2008

Abstract

Background

Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required.

Results

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.

Conclusion

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.