Transcriptome-Wide Identification and Validation of Reference Genes in Black Rockfish (Sebastes schlegelii)

The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique to analyze gene expression levels. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies on fishes adopted reference genes that were commonly used in mammals without validation. In this study, we utilized 89 transcriptome datasets covering early developmental stages and different adult tissues, and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii. Finally, 121 candidate reference genes were identified based on four criteria. Eight candidates (METAP2, BTF3L4, EIF5A1, TCTP, UBC, PAIRB, RAB10, and DLD) and four commonly used reference genes in mammals (TUBA, ACTB, GAPDH, RPL17) were selected for validation via qRT-PCR and four statistical analysis methods (delta-Ct, Best-Keeper, geNorm, and NormFinder). The results indicated that when the black rockfish are cultured in a general condition, the eight candidate reference genes are more stable than traditional reference genes in mammals, and RAB10, EIF5A1, PAIRB and BTF3L4 are the best reference genes in rockfish. This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish, and lay an important foundation for gene expression analysis in teleost.