Reference Genes Standardization for RT-qPCR of Abalone Tissues

The red abalone Haliotis rufescens is an important aquaculture species in Baja California, México. However, there are few studies related to gene expression in abalone gonadal tissues. Therefore, the main goal of this study was to obtain housekeeping genes (HKGs) that allow proper quantification in (reverse-transcriptase, quantitative PCR) RT-qPCR, since they have little o no change on their expression level regardless of the experimental treatment to which the biological sample is subjected or the development stage of the cell or tissue. Nine genes (Actb, Bglu, Tubb, Cy, Gapdh, Hprti, Rpl5, Sdha and Ubc) were validated in different tissues of red abalone (gill, cephalic tentacle and gonad). Four-fold serial dilutions of cDNA (from 25 ng µL^-1 to 0.39 ng µL^-1) were used to prepare the standard curve, which showed gene efficiencies between 0.95 and 0.99 with R² = 0.99. geNorm and NormFinder analysis indicated that Rpl5 and Cy were the most stable genes for all tissues. However, Rpl5 and Tubb were the most stable genes for gonads by geNorm, while Sdha and HprtI were the best using NormFinder. In addition, the Rpl5 variability analysis in abalone gonadal tissue indicated a high repeatability with a variation coefficient within groups of ≤ 0.50% and between groups of ≤ 1.89%. Based on these results, Rpl5 was the most appropriate HKG to be used as internal control in expression studies of gonadal tissue of red abalone.