Expression Stability of GAPDH as a Reference Gene in Fat Deposition Studies in Broiler Chickens: A Systematic Review

Background: Reliable normalization of gene expression data is essential for accurate interpretation of molecular mechanisms underlying fat deposition in broiler chickens. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is commonly used as a reference gene; however, its stability remains uncertain in adipose tissues. Methods: A systematic review was conducted, integrating evidence from transcriptomic and quantitative PCR studies. Relevant studies were identified through structured searches, citation tracking, and relevance screening, with emphasis on tissue specificity, developmental stage, and physiological variation. Results: GAPDH expression showed considerable variability across tissues and experimental conditions, particularly in adipose tissues where metabolic activity influences transcriptional regulation. In contrast, alternative reference genes, including 18S rRNA, PPIA, RPL19, and α-tubulin, demonstrated greater stability across conditions. Studies consistently reported that failure to validate reference genes introduces bias in gene expression analysis and may compromise interpretation of adipogenesis and lipid metabolism pathways. Conclusions: GAPDH is not a universally stable reference gene for adipose tissue studies in broiler chickens. Therefore, validation of reference genes is essential, and the use of multiple stable genes is recommended to ensure accurate normalization and reproducibility in poultry genomics research.