Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822)

•HKGs stability was tested in gill, head and gonad/digestive gland of red abalone.•RPL5 and CY were the most stable genes considering all tissues.•In gonad, the best genes were RPL5/TUBB with geNorm and SDHA/HPRTI with NormFinder.•Maximum values of intra and inter-assay variability were 0.50% and 1.89%.•In three tissues geNorm suggests to use two HKGs as normalization factor.

The red abalone Haliotis rufescens is one of the most important species for aquaculture in Baja California, México, and despite this, few gene expression studies have been done in tissues such as gill, head and gonad. For this purpose, reverse transcription and quantitative real time PCR (RT-qPCR) is a powerful tool for gene expression evaluation. For a reliable analysis, however, it is necessary to select and validate housekeeping genes that allow proper transcription quantification. Stability of nine housekeeping genes (ACTB, BGLU, TUBB, CY, GAPDH, HPRTI, RPL5, SDHA and UBC) was evaluated in different tissues of red abalone (gill, head and gonad/digestive gland). Four-fold serial dilutions of cDNA (from 25 ng μL− 1 to 0.39 ng μL− 1) were used to prepare the standard curve, and it showed gene efficiencies between 0.95 and 0.99, with R2 = 0.99. geNorm and NormFinder analysis showed that RPL5 and CY were the most stable genes considering all tissues, whereas in gill HPRTI and BGLU were most stable. In gonad/digestive gland, RPL5 and TUBB were the most stable genes with geNorm, while SDHA and HPRTI were the best using NormFinder. Similarly, in head the best genes were RPL5 and UBC with geNorm, and GAPDH and CY with NormFinder. The technical variability analysis with RPL5 and abalone gonad/digestive gland tissue indicated a high repeatability with a variation coefficient within groups ≤ 0.56% and between groups ≤ 1.89%. These results will help us for further research in reproduction, thermoregulation and endocrinology in red abalone.