Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells.

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.

Establishment of a Steatosis Model in LMH Cells, Chicken Embryo Hepatocytes, and Liver Tissues Based on a Mixture of Sodium Oleate and Palmitic Acid.

Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis.