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

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Abstract

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.

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Abbreviations

METAP2:

Methionine aminopeptidase 2

BTF3L4:

Transcription factor BTF3 homolog 4

EIF5A1:

Eukaryotic translation initiation factor 5A-1

TCTP:

Translationally-controlled tumor protein

UBC:

Ubiquitin C

PAIRB:

Plasminogen activator inhibitor 1 RNA-binding protein

RAB10:

Ras-related protein Rab-10

DLD:

Dihydrolipoamide dehydrogenase

TUBA:

α-Tubulin

RPL17:

Ribosomal protein L17

ACTB:

β-actin

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

qRT-PCR:

Quantitative reverse transcription PCR

TPM:

Transcripts per million

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Acknowledgement

This study was supported by the National Key R&D Program of China (No. 2018YFD0900101).

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Authors and Affiliations

  1. MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China

    Chaofan Jin, Weihao Song, Mengya Wang, Jie Qi, Quanqi Zhang & Yan He

  2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China

    Quanqi Zhang

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  1. Chaofan Jin
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  2. Weihao Song
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  4. Jie Qi
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Correspondence to Yan He.

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Jin, C., Song, W., Wang, M. et al. Transcriptome-Wide Identification and Validation of Reference Genes in Black Rockfish (Sebastes schlegelii). J. Ocean Univ. China 20, 654–660 (2021). https://doi.org/10.1007/s11802-021-4588-4

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  • DOI: https://doi.org/10.1007/s11802-021-4588-4

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