Quail Study Maps Stable Reference Genes for Thermal Stress qPCR

Researchers at the ICAR-Central Avian Research Institute in Bareilly, India, have identified tissue-specific reference genes that maintain stable expression in Japanese quail embryos subjected to embryonic thermal conditioning. Published in Biological Research, the study addresses a critical methodological gap in avian developmental physiology by validating internal controls for quantitative real-time PCR, or qPCR, under heat stress conditions. The findings provide a robust framework for normalizing gene expression data in studies exploring how early thermal exposure influences embryonic development and subsequent heat resilience in poultry.

The reliability of qPCR data hinges on the use of stable reference genes for normalization, yet the expression stability of these genes can vary significantly across different tissues and physiological states. In the context of Japanese quail, a model organism widely used for embryology and stress physiology research, prior information regarding suitable reference genes during embryonic heat stress was limited. This study systematically evaluated eight candidate reference genes to determine their suitability for normalizing target gene expression in liver, intestine, heart, and brain tissues under specific thermal conditioning protocols.

The experimental design involved 200 fertilized Japanese quail eggs, which were divided into three groups: a control group incubated under standard conditions, and two treatment groups subjected to embryonic thermal conditioning during early (days 6–8) and late (days 12–14) embryonic development. In the treatment groups, eggs were exposed to a temperature of 39.5 °C for 10 hours daily while maintaining a constant relative humidity of 55%. After the conditioning period, four embryos were randomly selected from each group, and tissue samples from the liver, intestine, heart, and brain were collected for RNA isolation and subsequent gene expression analysis.

The study assessed the stability of eight candidate reference genes: 18 S rRNA, β-ACTIN, 28 S rRNA, HPRT1, GAPDH, PGK1, RPS8, and TBP. These genes were selected based on their frequent use in avian gene expression studies and their roles in key cellular functions, including transcription, protein synthesis, cytoskeletal maintenance, metabolism, and nucleotide recycling. To ensure a comprehensive evaluation, the researchers employed five different statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder, which provides a comprehensive gene stability ranking by combining the results of the other four methods.

In liver tissue, the analysis revealed that HPRT1, GAPDH, and RPS8 emerged as the most stable reference genes across the various algorithms. Specifically, geNorm identified GAPDH, RPS8, and HPRT1 as having the lowest variability, while NormFinder ranked HPRT1 and GAPDH with the highest stability. BestKeeper similarly highlighted RPS8 and HPRT1 as reliable controls. In contrast, TBP and PGK1 were found to be the least stable genes in the liver, indicating that their expression levels fluctuated more significantly under thermal conditioning conditions.

For intestinal tissue, the stability rankings differed from those observed in the liver. PGK1, RPS8, and HPRT1 were identified as the most stable reference genes, with PGK1 consistently ranking at the top in several analyses, including RefFinder and NormFinder. The Delta Ct method also identified PGK1, HPRT1, and RPS8 as the most stable, while 28 S and 18 S rRNA displayed the highest variability. These findings suggest that PGK1, RPS8, and HPRT1 are well-suited as internal controls for qPCR normalization in the intestines of quail embryos undergoing thermal conditioning.

In heart tissue, TBP and HPRT1 were identified as the most reliable reference genes for normalization. The Delta Ct, geNorm, and NormFinder analyses consistently ranked TBP, HPRT1, and GAPDH as the most stable genes, with TBP often appearing as the top candidate. However, BestKeeper ranked β-ACTIN and PGK1 as the most stable, highlighting the importance of using multiple statistical tools to reach a consensus. The 28 S rRNA gene was consistently identified as the least stable reference gene in heart tissue across most algorithms, reinforcing the need for empirical validation of traditional markers.

Brain tissue analysis showed that HPRT1 and GAPDH were the top stable genes across most algorithms, consistent with patterns observed in other tissues. The Delta Ct method ranked HPRT1, GAPDH, and 18 S as the most stable, while geNorm and NormFinder identified HPRT1 and GAPDH as having the lowest stability values. BestKeeper differed slightly, ranking β-ACTIN and HPRT1 as the most stable, but still identified TBP and PGK1 as the least stable. Overall, HPRT1 and GAPDH emerged as reliable internal reference markers for qPCR normalization in quail brain tissue under embryonic thermal conditions.

When evaluating all tissues combined, TBP and RPS8 emerged as the most suitable gene set for normalization. These two genes were consistently ranked among the most stable via Delta Ct, geNorm, and NormFinder, while 28 S rRNA was the least stable across all methods. The RefFinder analysis, which provides a comprehensive consensus ranking, suggested that TBP, followed by RPS8, was the most stable gene for normalizing gene expression data in experiments involving embryonic thermal conditioning. This combined analysis underscores the tissue-specific nature of reference gene stability and the necessity of selecting appropriate controls based on the specific tissue being studied.

The study highlights that no single reference gene demonstrates consistent stability across all tissue types, emphasizing the critical need for tissue-specific validation. The consistent instability of 28 S and 18 S rRNA genes across multiple tissues and algorithms suggests that these traditionally used rRNA genes may not be ideal internal controls in thermally challenged embryos. The results provide a robust foundation for future qPCR-based research on gene expression in avian embryonic tissues, ensuring that data interpretation is accurate and reproducible.

The findings align with previous studies in other poultry species, such as chickens and ducks, where similar patterns of gene stability were observed under heat stress or different physiological conditions. For instance, previous research in chickens has also identified the instability of β-ACTIN and rRNA genes under stress, supporting the current findings in quail. The study also notes that while some genes like GAPDH were stable in certain tissues, their stability can vary depending on the specific experimental context, such as the type of stressor or developmental stage.

The authors conclude that HPRT1, RPS8, and GAPDH may be recommended for liver tissue; PGK1, RPS8, and HPRT1 for intestine; TBP, HPRT1, and GAPDH for heart; and HPRT1 and GAPDH for brain tissue. Overall, TBP and RPS8 are recommended as stable genes for qPCR normalization under embryonic thermal conditions in quails across different tissues. These validated reference genes provide a reliable foundation for accurate and reproducible qPCR-based gene expression studies in thermally conditioned quail embryos, contributing to the broader understanding of avian developmental and stress physiology.

Beyond its utility in poultry production for meat and eggs, the Japanese quail serves as an advantageous avian model for developmental stress physiology due to its rapid maturation within five to six weeks, year-round breeding capacity, and fully sequenced genome. Because avian embryos develop inside eggs outside the maternal body, environmental conditions such as temperature, noise, and light can be precisely modulated during critical incubation windows. In this investigation, the researchers selected two hundred eggs of the CARIUTTAM variety from parents of similar age, applying strict physical criteria that retained only medium-sized eggs of standard shape while excluding cracked, misshapen, oversized, or undersized specimens. Control eggs were maintained continuously under standard incubation parameters of 37.8 degrees Celsius and 55 percent relative humidity. To preserve methodological rigor, researchers maintained blinding throughout the incubation timeline before harvesting the four target tissues and storing them in tissue preservation reagent at minus twenty degrees Celsius.

The laboratory workflow incorporated rigorous biochemical validation steps to ensure template integrity prior to quantification. Harvested liver, intestine, heart, and brain samples were rinsed with nuclease-free water, triturated in TRIzol reagent, and checked for RNA yield and purity using spectrophotometry alongside agarose gel electrophoresis to confirm nucleic acid integrity. For cDNA synthesis, one microgram of total RNA was reverse-transcribed using random hexamers and reverse transcriptase during a thirty-minute incubation at 42 degrees Celsius, terminated by heat inactivation at 95 degrees Celsius. Primer sets for the eight candidate genes underwent temperature gradient optimization on a thermal cycler, with resulting amplicons verified for expected size and specificity through 1.8 percent agarose gel electrophoresis against a 50-base-pair molecular weight ladder in tris-acetate-EDTA buffer.

Real-time quantitative amplification was conducted in triplicate across 96-well microplates using SYBR Green master mix, encompassing an initial fifteen-minute denaturation followed by forty amplification cycles and melting curve analysis from 65 to 95 degrees Celsius to confirm single-peak amplicon specificity. The evaluation drew upon distinct algorithmic principles to assess expression behavior. The geNorm program evaluated pairwise expression ratios, designating candidates with lower M values as more stable against an optimal threshold of 1.0 or lower. NormFinder estimated stability values by directly calculating both intra-group and inter-group variations across conditions, whereas BestKeeper calculated standard deviations from raw crossing points, identifying acceptable internal controls while excluding candidates with standard deviations exceeding 1.0. Finally, RefFinder assigned weights to individual program rankings and computed their geometric mean to deliver a balanced consensus hierarchy.

These methodological findings align with broader industry efforts to enhance thermal resilience in poultry production amid climate change and rising temperatures. Prior research in chickens demonstrated that applying thermal conditioning for three to twelve hours daily during late embryonic incubation fostered thermal adaptation without adversely impacting hatchability or post-hatch performance. By systematically identifying reliable internal standards in Japanese quail, the authors adhered to international reporting recommendations that emphasize validating reference candidates across distinct tissues and experimental interventions rather than assuming uniform stability across avian species.

Subject of Research: Tissue-specific reference gene stability for qPCR in thermally conditioned quail embryos

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

Article References: Ansari, M. M., Martina, K., Kolluri, G., Tyagi, J. S., Rokade, J., Bhutekar, P., Naim, A., Gangwar, M., & Ahmad, S. F. (2026). Tissue-specific reference gene stability for qPCR in thermally conditioned quail embryos. Biological Research, 59(1), Article 51. https://doi.org/10.1186/s40659-026-00714-w

Image Credits: AI Generated

DOI: 10.1186/s40659-026-00714-w

Keywords: Tissue-specific, reference, gene, stability, qPCR, thermally, conditioned, quail, embryos, scientific research