Selection and evaluation of dual reference genes in silkworm (Bombyx mori ) based on transcriptome data

Objective The stability of reference genes is critical for qPCR data accuracy, especially in insect developmental studies with stage-specific transcriptional variation. This study aimed to identify stable reference genes for Bombyx mori’s entire developmental cycle and validate their utility in target gene normalization. Methods Transcriptomic data covering the entire developmental cycle of B. mori were screened for stable reference genes using principal component analysis (PCA) and Venn analysis. A total of 125 highly expressed and stable candidate genes were identified and nine candidate reference genes were selected based on the coefficient of variation (CV), with actin included as a traditional control. The stability of these nine genes was systematically evaluated using three algorithms: Delta CT, BestKeeper, and NormFinder, while the geNorm algorithm determined the optimal reference gene pair. The performance of the identified dual-reference system was validated by normalizing seven target genes and comparing results with normalization using actin alone. Results The optimal reference genes varied across the developmental stages of B. mori. Although no single gene was universally stable during all stages, the geNorm algorithm identified two genes (006219 and 000526) as the most stable pair for normalization throughout the entire developmental cycle. Normalization of seven target genes using this dual-reference system yielded consistent and reliable expression trends, whereas normalization with actin alone resulted in significant expression biases across developmental stages. Conclusions The dual-reference gene system effectively minimizes technical errors, enhancing the accuracy and reproducibility of qPCR data. Actin is not a universally stable reference gene for B. mori, and this study provides a reliable set of reference genes for qPCRbased gene expression analysis in B. mori and that serves as a valuable reference for similar studies on other insects. Further validation of this dual-reference system under diverse experimental conditions will contribute to advanced molecular research in insects.