Reference genes for gene expression studies in pacu (Piaractus mesopotamicus) raised in different culture systems

Abstract

Reference genes (RGs) are crucial for reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis as they allow accounting for uncontrolled environmental variations in gene expression results. Thus, prior reliability assessment of RGs is essential. In this study, candidate RGs were evaluated to investigate the gene expression responses of Piaractus mesopotamicus (pacu) reared in biofloc technology (BFT) and recirculating aquaculture systems (RAS). Samples from skeletal muscle (n = 9 BFT and 9 RAS) and kidney (n = 9 BFT and 9 RAS) were collected from juvenile pacu weighing 68 ± 4.43 g. The stability of three candidate RGs (GAPDH, ACTB, and 18S) was evaluated using GeNorm, BestKeeper, and NormFinder software. GAPDH was the least stable gene in pacu, due to high variability within and between BFT and RAS. In kidney tissue, ACTB was identified as the most stable gene by all three software. In skeletal muscle, NormFinder and BestKeeper classified 18S as the most stable gene, while GeNorm suggested a combination of 18S and ACTB. This is the first investigation on reference genes for qPCR in juvenile pacu tissues, and the findings will provide valuable support for gene expression studies in this species. Among the three candidate RGs tested in this study, ACTB and 18S genes are recommended for qPCR in kidney and muscle tissues of pacu, respectively, while GAPDH was deemed the least suitable. The observed differences in the expression of candidate genes among BFT and RAS and tissues emphasize the importance of appropriate RG selection prior to conducting RT-qPCR experiments.