Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Abstract

Background: The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique that relies on the reference gene for gene expression normalization. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies of fishes adopted reference genes that were commonly used in mammals without validation. Results: In this study, we utilized 89 transcriptome datasets covering early developmental stages and 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 (TUBA, ACTB, GAPDH, RPL17) in mammals were selected for validation via qRT- PCR and four statistical methods (delta-Ct, BestKeeper, geNorm, and NormFinder). The results revealed that the candidate reference genes we recommended are more stable than traditionally used ones. Conclusions: 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.