Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues

Area of Science:

• Animal Genomics
• Molecular Biology
• Gene Expression Analysis

Background:

• Accurate normalization is essential for quantitative PCR (qPCR) in livestock research.
• The chicken, a major livestock species, lacks established reference genes for growth and fattening studies.
• Previous transcriptomic analyses in chickens highlight the need for robust qPCR normalization strategies.

Purpose of the Study:

• To identify and validate stable reference genes for qPCR normalization in chicken tissues.
• To evaluate candidate reference genes using multiple statistical algorithms.
• To provide reliable normalization factors for gene expression studies in chicken growth and metabolism.

Main Methods:

• Evaluation of five candidate reference genes (B2M, RPL32, SDHA, TBP, YWHAZ) in pectoralis major, biceps femoris, liver, and abdominal fat.
• Application of four statistical methods: geNorm, NormFinder, Coefficient of Variation (CV), and BestKeeper.
• Comprehensive ranking and validation using the RefFinder tool and a target gene (SCD).

Main Results:

• YWHAZ and TBP identified as the best combination for biceps femoris and liver.
• YWHAZ and RPL32 recommended for pectoralis major.
• RPL32 and B2M suitable for abdominal fat and across-tissue analyses.
• Validation using SCD confirmed the reliability of the selected reference genes.

Conclusions:

• Established reliable reference gene sets for accurate qPCR normalization in various chicken tissues.
• The findings facilitate precise gene expression quantification in chicken growth, fattening, and metabolic studies.
• Provides a foundation for future transcriptomic and molecular analyses in poultry research.