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transcriptome-wide Identification and Validation of Reference Genes in Black Rockfish(Sebastes schlegelii)

Journal of Ocean University of China 2021年第3期20卷 654-660页

作者： JIN Chaofan SONG Weihao WANG Mengya QI Jie ZHANG Quanqi HE Yan MOE Key Laboratory of Marine Genetics and Breeding College of Marine Life SciencesOcean University of ChinaQingdao 266003China Laboratory for Marine Fisheries Science and Food Production Processes Qingdao National Laboratory for Marine Science and TechnologyQingdao 266237China

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 transcrip-tome 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 mam-mals(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.

关键词： reference genes black rockfish transcriptome-wide CV value qRT-PCR

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Epitranscriptomic technologies and analyses

引用

Science China(Life Sciences) 2020年第4期63卷 501-515页

作者： Xiaoyu Li Qiao-Xia Liang Jin-Ran Lin Jinying Peng Jian-Hua Yang Chengqi Yi Yang Yu Qiangfeng Cliff Zhang Ke-Ren Zhou State Key Laboratory of Protein and Plant Gene Research School of Life SciencesPeking UniversityBeijing 100871China MOE Key Laboratory of Gene Function and Regulation State Key Laboratory for BiocontrolSchool of Life SciencesSun Yat-Sen UniversityGuangzhou 510275China The Fifth Affiliated Hospital Sun Yat-Sen UniversityZhuhai 519000China Department of Chemical Biology and Synthetic and Functional Biomolecules Center College of Chemistry and Molecular EngineeringPeking UniversityBeijing 100871China Key Laboratory of RNA Biology CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101 China MOE Key Laboratory of Bioinformatics Beijing Advanced Innovation Center for Structural BiologyCenter for Synthetic and Systems BiologyTsinghua-Peking Joint Center for Life SciencesSchool of Life SciencesTsinghua UniversityBeijing 100084China

RNA can interact with RNA-binding proteins(RBPs),mRNA,or other non-coding RNAs(ncRNAs)to form complex regulatory networks.High-throughput CLIP-seq,degradome-seq,and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions.However,assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs)remains technically challenging.Chemical modifications to mRNA also play important roles in regulating gene expression.Investigation of the functional roles of these modifications relies highly on the detection methods used.RNA structure is also critical at nearly every step of the RNA life cycle.In this review,we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs.We also summarize methods used to detect RNA modifications and to probe RNA structure.

关键词： ncRNA bioinformatics CLIP-seq RNA modification quantification and locus-specific detection methods transcriptome-wide sequencing technologies RNA structuromes RNA structure probing methods

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