Stable Reference Genes for RT ‐ qPCR in House Dust Mite Dermatophagoides farinae (Acari: Pyroglyphidae) at Different Developmental Stages

ABSTRACT To investigate the growth and development mechanisms in Dermatophagoides farinae , it is essential to understand its gene expression profiles across different life stages, which are typically assessed by transcriptional profiling based on RT‐qPCR. Suitable reference genes are vital for ensuring reliable and accurate results; however, none have been established for this species across different developmental stages. Here, we analyzed expression profiles of ten frequently used reference genes ( RPL18 , RPS18 , RPS 9, EF1A , RPS28 , Actin , V‐ATPaseB , GAPDH , RPL32 , and α‐tubulin ) to determine their stability across different developmental stages of D. farinae . Four algorithms (GeNorm, NormFinder, BestKeeper, and ΔCt) were employed to examine gene stability, and their results were subsequently integrated by RefFinder to yield a comprehensive assessment. The results indicated that RPL18 and RPS28 were the two top‐ranked stable reference genes, whereas α‐tubulin and RPL32 showed the lowest stability. To confirm the robustness of the chosen reference genes, we assessed cytochrome P450 3A8 expression at developmental stages of D. farinae using three independent biological replicates and three technical replicates per stage. Normalization against RPL18 and RPS28 , individually or combined, produced expression patterns consistent with previously reported developmental trends of cytochrome P450 genes in mites and insects, namely higher expression in larvae and protonymphs, lower expression in later stages, and minimal expression observed in eggs. In contrast, unstable reference genes yielded inconsistent results, thereby confirming our findings. In conclusion, this study identifies two highly stable reference genes that will provide a robust foundation for precise gene expression normalization and functional studies in D. farinae .