Screening and Stability Analysis of Reference Genes in Pastor roseus

Background/Objectives: Optimal reference genes for normalizing RT-qPCR data depend on the species, treatments, developmental stages, and other conditions. Pastor roseus is a long-distance migratory bird with potential applications in locust biological control. This study applied reverse transcription quantitative PCR (RT-qPCR) to evaluate the expression stability of six genes (RPS2, ACTB, B2M, SDHA, UBE2G2, and RPL4) in blood samples from female, male, and nestling P. roseus. Methods: An integrated analysis of the expression stability of six reference genes was performed using three statistical algorithms: GeNorm, BestKeeper, and NormFinder. Results: The results showed that SDHA, ACTB, and B2M exhibited the highest expression stability among the candidate reference genes. The optimal number of reference genes was two, as determined by a pairwise variation analysis using GeNorm. Subsequent comprehensive validation using RefFinder identified SDHA/ACTB as the optimal reference gene pair for normalizing gene expression data for P. roseus. Conclusions: These findings establish a robust foundation for ensuring data accuracy in functional genomic studies of P. roseus.