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

Area of Science:

• Avian developmental biology
• Molecular biology
• Gene expression analysis

Background:

• Quantitative real-time PCR (qPCR) requires stable reference genes for accurate transcript quantification.
• Reference gene stability can vary significantly across different tissues and experimental conditions.

Purpose of the Study:

• To evaluate the expression stability of eight candidate reference genes in Japanese quail embryos subjected to embryonic thermal conditioning (ETC).
• To identify the most reliable reference genes for normalization in qPCR studies under heat stress in quail embryos.

Main Methods:

• Assessed expression levels of eight candidate reference genes (18S rRNA, β-ACTIN, 28S rRNA, HPRT1, GAPDH, PGK1, RPS8, TBP) in liver, intestine, brain, and heart tissues.
• Utilized five algorithms (geNorm, NormFinder, BestKeeper, Delta Ct, RefFinder) to assess gene expression stability.
• Employed embryonic thermal conditioning (ETC) involving heat exposure (39.5°C) on specific embryonic days.

Main Results:

• Gene expression stability varied across tissues and algorithms.
• HPRT1, GAPDH, and RPS8 were most stable in liver; PGK1, RPS8, and HPRT1 in intestine.
• TBP and HPRT1 were most reliable in heart; HPRT1 and GAPDH in brain.
• TBP and RPS8 were the most suitable gene set for normalization across all examined tissues.

Conclusions:

• This study provides essential guidelines for selecting appropriate reference genes in quail embryos under heat stress.
• The findings enhance the reliability and accuracy of qPCR data in avian developmental and stress physiology research.