Tissue-specific reference gene stability for qPCR in thermally conditioned quail embryos

The reliability and accuracy of relative quantification of transcripts through quantitative real-time PCR (qPCR) is dependent on the use of stable reference genes for normalization. However, the expression stability of various reference genes may differ across the tissues and patho-physiological conditions. The present study investigated the stability of multiple candidate reference genes in Japanese quail (Coturnix japonica) embryos, subjected to embryonic thermal conditioning (ETC). Fertilized quail eggs (n = 200) were incubated under standard conditions and heat exposure (ETC) of incubated eggs was undertaken at 39.5 °C for 10 h daily in the embryonic days 6-8 and 12-14, maintaining constant humidity (55%). Four embryos were randomly selected from each group for experimentation. The expression levels of eight candidate reference genes i.e., 18 S rRNA, β-ACTIN, 28 S rRNA, HPRT1, GAPDH, PGK1, RPS8, and TBP were assessed across four tissues (liver, intestine, brain, and heart) under ETC. Gene expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder (Comprehensive gene stability). Variations in gene stability were evident across different tissues and algorithms. The HPRT1, GAPDH and RPS8 emerged as the most stable reference genes for qPCR analyses in liver tissue, making them suitable internal controls. Similarly, PGK1, RPS8, and HPRT1 genes exhibited higher expression stability in intestine. On the other hand, TBP and HPRT1 were identified as the most reliable reference genes for normalization in heart tissue, whereas HPRT1 and GAPDH were found to be the most reliable reference genes in brain tissue. On considering all tissues combined (liver, intestine, brain, and heart), TBP and RPS8 emerged as the most suitable gene set for normalization. This present study provides crucial insights into selecting appropriate reference genes for relative expression quantification studies in quail embryos under heat treatment (ETC), thereby improving the reliability and accuracy of qPCR data in avian developmental and stress physiology research.