Identification and validation of reference genes for RT-qPCR single-gene relative expression analysis in Taenia solium pre-adult stages

The larvae-to-adult development of the zoonotic parasitic tapeworm Taenia solium involves significant but often clinically overlooked events crucial in cestode biology. The early-adult stages can be studied in vitro, providing a valuable model to examine scolex evagination, strobilation, and worm development. Without a stage-specific transcriptome, postgenomic data exploration followed by single-gene relative expression analysis using RT-qPCR (reverse transcription-quantitative PCR) are effective strategies to study gene regulation during parasite development. However, achieving accurate comparisons with this approach requires the validation of an endogenous reference gene (RG). To address this, we analyzed the expression stability of 17 candidate reference genes (RGs), representing various biological processes, in the context of the in vitro-induced early adult stages of T. solium larvae (cysts). RT-qPCR of the candidate RGs was performed on different stages, defined by distinct morphology in culture, and gene expression stability was comprehensively analyzed using the RefFinder tool. Genes pgk1, bact1, mapk3, tbp, rpl13, and cox1 were ranked as the most stable and were used to normalize the expression of h2b and wnt11a, which are involved in proliferation and strobilation processes in parasitic tapeworms. This study represents the first attempt to identify reliable normalization standards for transcript analysis in the genus Taenia.