Screening and Stability Analysis of Reference Genes in Pastor roseus

<b>Background/Objectives:</b> Optimal reference genes for normalizing RT-qPCR data depend on the species, treatments, developmental stages, and other conditions. <i>Pastor roseus</i> 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 (<i>RPS2</i>, <i>ACTB</i>, <i>B2M</i>, <i>SDHA</i>, <i>UBE2G2</i>, and <i>RPL4</i>) in blood samples from female, male, and nestling <i>P. roseus</i>. <b>Methods:</b> An integrated analysis of the expression stability of six reference genes was performed using three statistical algorithms: GeNorm, BestKeeper, and NormFinder. <b>Results:</b> The results showed that <i>SDHA</i>, <i>ACTB</i>, and <i>B2M</i> 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 <i>SDHA</i>/<i>ACTB</i> as the optimal reference gene pair for normalizing gene expression data for <i>P. roseus</i>. <b>Conclusions:</b> These findings establish a robust foundation for ensuring data accuracy in functional genomic studies of <i>P. roseus</i>.