Determination of Stable Reference Genes for Gene Expression Analysis in Black Rockfish (Sebastes schlegeli) Under Hypoxia Stress.

BACKGROUND: Hypoxia triggers stress, leading to significant alterations in gene expression patterns, which in turn affect fish's growth and development. Real-time quantitative PCR (RT-qPCR) is a pivotal technique for assessing changes in gene expression. However, its accuracy is highly contingent upon the stable expression of reference genes. Ribosomal RNA (18s), β-actin (actb), elongation factor 1-α (ef1a), α tubulin (tuba), and ribosomal protein L17 (rpl17) are the widely used reference genes, but their expression stability in the tissues of black rockfish under hypoxic conditions remains unclear. METHODS: The expression of genes was detected by RT-qPCR and the stability was assessed by Delta Ct, geNorm, NormFinder, and BestKeeper algorithms. RESULTS: Results showed that tuba exhibited stable expression in liver, heart, gill tissues under normoxic conditions, and in the liver and head kidney under hypoxic conditions. Ef1a was identified as the most stably expressed gene in gill tissue under hypoxia. For hypoxic heart studies, rpl17 and tuba were recommended as reference genes. 18s showed high stability in spleen tissue under hypoxic conditions. Actb was the most stably expressed gene in spleen and head kidney tissues under normoxic conditions. CONCLUSIONS: The identified reference genes exhibited tissue-specific stability, and it was necessary to select appropriate reference genes based on the specific tissue type for gene expression studies under hypoxic conditions. These findings help in enhancing the accuracy of gene expression analysis in the mechanism of hypoxia for black rockfish.