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Abstract：
The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.
摘要：
肝脏在家禽脂质合成和代谢中起着至关重要的作用。为了更有效地研究功能基因，在鸡肝中筛选可靠的参考基因至关重要，包括女性,男性,胚胎,以及Leghorn雄性肝癌（LMH）细胞系。传统的参考基因筛选涉及选择常用的管家基因（HKG）进行RT-qPCR实验，并使用不同的算法来鉴定最稳定的基因。然而,这种方法在从一小批HKG中选择最佳参考基因时受到限制。高通量测序技术可以为这种限制提供解决方案。这项研究旨在通过利用鸡肝和LMH细胞的多个已发表的RNA-seq数据来鉴定最一致表达的基因。随后，使用RT-qPCR，与先前验证的稳定家禽肝脏表达的参考基因和常用的HKG进行比较，评估了新鉴定的参考基因的稳定性。结果表明，雌雄肝脏稳定表达基因（如LSM14A和CDC40）具有较高的相似性。在胚胎肝脏中，最佳的新内参基因是SUDS3,TRIM33和ERAL1.对于LMH细胞，最佳的新参考基因是ALDH9A1，UGGT1和C21H1orf174。然而,值得注意的是，大多数HKG在多个样本中没有表现出稳定的表达，表明在不同条件下的潜在不稳定性。此外，RT-qPCR实验证明，从RNA-seq数据中鉴定出的稳定表达基因优于常用的HKG和某些经过验证的家禽肝脏特异性参考基因。总的来说，这项研究成功地鉴定了新的稳定的内参基因在鸡肝和LMH细胞使用RNA-seq数据，为研究人员在不同情况下的RT-qPCR提供更广泛的参考基因选择。
