Morphological and transcriptomic analyses of the developing chicken chorioallantoic membrane and the validation of qPCR reference genes

Background The chorioallantoic membrane (CAM) is a popular model for transplantation and angiogenesis studies. However, unlike the embryo itself, the CAM lacks standardized morphological and molecular benchmarks, making it difficult to accurately quantify host reactions. We analyzed the CAM's structure and gene expression from embryonic day 8 (d8) to d18. Results Morphological analysis showed that the fundamental layer structure of the CAM is established by d8. To ensure accurate molecular analysis, we used RNA-seq and qPCR to search for stable reference genes. We identified RPL5 as the suitable reference gene for the CAM. Crucially, we found that using the common reference gene GAPDH can distort quantitative interpretations of developmental expression profiles, whereas RPL5 provides a stable reference baseline that minimalizes normalization bias. Conclusions This study establishes a morphological and transcriptomic characterization of CAM development while establishing a validated reference gene candidate (RPL5) for quantitative analysis. Our findings offer a critical foundation for reliable and reproducible quantitative analysis in various CAM-based research models.