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.H...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.展开更多
文摘为了获取生殖细胞移植的不育受体,本研究使用白消安(1,4-丁二醇二甲磺酸酯)对二龄半红鳍东方鲀(Takifugu rubripes)进行处理,使用40 mg/kg白消安注射处理两次后,进行了对照组、注射组的性腺取样和转录组测序,分析了白消安的处理效果和白消安对性腺基因表达的影响。研究表明:40 mg/kg白消安其对精巢的处理效果显著,生精小管中的生殖细胞大量减少;对卵巢的处理效果不佳,实验组与对照组的卵巢在组织形态上无显著差异。性腺转录组的分析结果显示,与注射二甲基亚砜(Dimethyl sulfoxide,DMSO)溶剂的对照组相比,经过白消安处理后,精巢组中获得差异表达基因576个,卵巢组中获得差异表达基因74个,白消安处理对精巢基因的转录水平影响更大。差异表达基因的GO(Gene Ontology)富集分析显示,氧化还原酶活性、单加氧酶活性、双加氧酶活性等氧化代谢相关条目被显著富集;KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析显示,氧化磷酸化、核苷酸切除修复、范可尼贫血、同源重组、DNA复制和错配修复等通路被显著富集。GSEA(Gene set enrichment analysis)结果显示,错配修复、范可尼贫血、同源重组等通路被上调,DNA复制和核苷酸切除修复通路下调。研究结果表明,白消安处理导致了雄性受体的内源生殖细胞数量减少,并使精巢处于DNA损伤和氧化应激的状态。不同性腺组织对白消安的不同反应可能与其细胞类型对氧化应激和DNA损伤的抵抗有关。
基金This study was supported by the National Key R&D Program of China(No.2018YFD0900101).
文摘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.
