Screening and Evaluating Reference Genes for Quantitative Real-Time PCR in Striped Jack (Pseudocaranx dentex)

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, often implemented to allow for the detection and quantitation of gene expression because of its high sensitivity, specificity, and reproducibility. However, reliable and comparable relative quantitative results using qRT-PCR require the application of appropriate reference genes in order to eliminate non-biological variations caused by initial RNA templates, efficiency of cDNA synthesis, and laboratory procedures. However, previous studies have reported that the stability of many reference genes may vary across species, tissue types, cell lines, developmental stages, and experimental treatments, yielding inaccurate or incorrect gene expression results. Therefore, the selection and validation of stable reference genes for different tissues from a specific species are especially important for obtaining accurate target gene expression results. The striped jack, Pseudocaranx dentex, belonging to the order Perciformes and family Carangidae, is a pelagic migratory fish with high nutritional value. This fish has already received extensive attention in global aquaculture production and is regarded as a candidate species for far-reaching marine aquaculture in China. Given this, there are currently a large number of fairly extensive molecular biology and genetics studies of P. dentex underway, which in turn have increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. However, few studies have evaluated the reference genes for this species. Thus, the objective of this study was to identify suitable reference genes in different tissues of P. dentex, in an effort to provide the necessary tools to support subsequent gene expression pattern analysis.We evaluated nine commonly used reference genes, including beta actin (β-actin), ribosomal protein L13 (RPL13), elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase (HPRT), peptidylprolyl isomerase A (PPIA), beta 2-microglobulin (β2M), beta tubulin (TUB), and serine/threonine-protein phosphatase 2A catalytic subunit (PP2A) using qRT-PCR analysis across various P. dentex tissues. These evaluations included the study of their expression stability across ten tissues, including the brain, gill, heart, intestine, kidney, liver, spleen, stomach, slow-twitch muscle, and fast-twitch muscle, from three adult individual