Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.

For relative quantification of gene expression the mRNA levels of target genes must be normalized. The most common normalization strategy is the utilization of internal reference genes. However, the stability of reference genes must be validated for the conditions of the study. The aim of the present work is to select the best reference genes in the bivalve mollusc Pecten maximus during the reproductive cycle from a set of seven candidates: actin (act), 18S ribosomal RNA (18S), cytochrome c oxidase subunit 1 (cox1), eukaryotic translation elongation factor 1 alpha (ef1a), glyceraldehyde-3-phosphate dehydrogenase (gapdh), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 7 (ndufa7), and 40S ribosomal protein SA (rpsa). In order to have samples at different stages of the reproductive cycle three specimens were sampled at approximately monthly intervals during a period of 7 months. Three different statistical models (geNorm, NormFinder and BestKeeper) have been used to analyze the results obtained by reverse transcription quantitative real time PCR (RT-qPCR). The most suitable reference genes were specific to each tissue. In P. maximus ovary ndufa7, rpsa and ef1a were the most stable genes. In P. maximus testis 18S, ndufa7 and gapdh were the best ranked reference genes.

► Reference gene selection for RT-qPCR in the mollusc Pecten maximus was investigated. ► Two tissues (ovary and testis) were analyzed during the reproductive cycle. ► The most suitable reference genes were specific to each tissue. ► ndufa7, rpsa and ef1a were the most stably expressed genes in the ovary. ► 18S rRNA, gapdh and ndufa7 were the best reference genes in the testis.