Evaluation and validation of reference genes for RT-qPCR analysis in different tissues of Echinococcus multilocularis-infected mice.

Echinococcosis, caused by the larval stage of the tapeworm Echinococcus species, threatens global public health and livestock economics. Currently, quantitative reverse transcription polymerase chain reaction (RT-qPCR) is essential for mRNA quantification, and its accuracy relies on stably expressed reference genes that must be validated under specific experimental conditions. In this study, we systematically evaluated the expression stability of twelve candidate reference genes in the liver, lung, and spleen of E. multilocularis-infected and healthy controls (n = 6 per group). Based on raw cycle threshold (Ct) values generated by RT-qPCR, the stability of each candidate gene was evaluated using both unstratified (samples from the same tissue were treated as a single cohort) and stratified (samples grouped by experimental condition) strategies. Then multiple algorithms including NormFinder, geNorm, BestKeeper, and Delta-Ct were employed to generate a comprehensive stability ranking of candidate genes. The validated optimal reference gene combinations were subsequently used to normalize gene expression via the comparative Ct (2-ΔΔCt) method. Our findings demonstrated that optimal reference genes were tissue-specific under E. multilocularis infection. For example, stratified analysis identified Actb as the most stable in both the liver (stability value = 0.16) and lung (stability value = 0.29), and Rpl13a (stability value = 0.10) in the spleen. Consequently, distinct reference gene combinations were validated and recommended respectively for liver, lung, and spleen, providing a reliable basis for accurate gene expression analysis by RT-qPCR. The reference sets will facilitate future investigations into echinococcosis, including disease pathogenesis, antiparasitic drug efficacy assessment, and therapeutic target discovery.