利用公开可用的RNA-seq数据来筛选理想的参考基因比传统方法更有效和准确。以前的研究已经确定了各种鸡组织中的最佳参考基因,但没有人特别关注输卵管(包括漏斗,大号,地峡,子宫,和阴道),这对鸡蛋生产至关重要。确定输卵管中稳定的参考基因对于改善基因表达水平的研究至关重要。这项研究调查了在鸡输卵管中具有一致表达模式的基因,包括个别输卵管组织和整个输卵管,通过利用多个RNA-seq数据集。筛选结果显示,在输卵管组织的每个片段中发现了100个新的内参基因,主要与细胞周期调控和RNA结合有关。此外,大多数管家基因(HKG)在不同样本中显示出不一致的表达水平,表明它们在不同条件下缺乏稳定性。与先前在鸡输卵管和常用的HKG中验证的稳定参考基因相比,评估了新鉴定的参考基因的稳定性,采用传统的参考基因筛选方法。HERPUD2,CSDE1,VPS35,PBRM1,LSM14A,和YWHAB被鉴定为输卵管不同部位的合适新参考基因。HERPUD2和YWHAB对于整个输卵管道的基因表达正常化是可靠的。此外,在DF1细胞中的过表达和干扰分析显示LSM14A和YWHAB在细胞增殖中起着至关重要的作用,强调了这些新参考基因对进一步研究的重要性。总的来说,这项研究扩大了在鸡输卵管和整个输卵管的不同节段的RT-qPCR实验中参考基因的选择。
Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken
oviduct, encompassing both individual
oviduct tract tissues and the entire
oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of
oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the
oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire
oviduct.