Identification of stable miRNA references for qRT-PCR normalization in ovine fasciolosis

Fasciola hepatica (F. hepatica) is a widely prevalent trematode responsible for parasitic infections and remains a major target for control interventions worldwide. Its ability to establish chronic infection relies on complex host-parasite interactions mediated by molecular mechanisms that regulate gene expression. Among these mechanisms, microRNAs (miRNAs), a class of non-coding RNAs, act as key post-transcriptional regulators and play important roles in immune modulation during infection. These molecules are increasingly investigated as potential diagnostic biomarkers and targets for novel vaccine strategies. However, accurate miRNA quantification by qRT-PCR requires stable endogenous normalizers to control for technical and biological variability, and such normalizers have not yet been validated for fasciolosis studies in ovine plasma. Therefore, this study aims to identify stable endogenous miRNA reference genes for RT-qPCR normalization in plasma samples from sheep experimentally infected with F. hepatica during the early stages of infection. We evaluated a panel of candidate miRNAs and ranked their expression stability using RefFinder, an integrative tool that combines several algorithms, including NormFinder, the ΔCt method, geNorm, and BestKeeper.

Stability analysis identified hsa-let-7c-5p, bta-let-7f, and oar-miR-16b as the most stable miRNAs across all conditions. These miRNAs showed consistent expression across samples and time points, indicating minimal susceptibility to infection-related biological variation or technical bias. They are therefore suitable endogenous normalizers for plasma miRNA quantification in early fasciolosis. The geometric mean of these three miRNAs was employed for normalization to enable accurate and reproducible relative quantification of three target miRNAs whose expression patterns are associated with distinct stages of Fasciola infection. Temporal profiling revealed an early upregulation of oar‑miR‑133‑5p, a later peak of oar‑miR‑3957‑5p, and a decreasing trend of fhe‑miR‑novel‑11‑5p. Because we obtained these expression patterns after normalization with rigorously validated stable references, they are more likely to reflect true biological dynamics rather than technical variability. Together, these findings support the stage-specific diagnostic potential of the target miRNAs and further implicate their involvement in host-parasite interactions during infection.

These findings highlight the value of validated miRNAs as endogenous reference genes of plasma-based studies in ovine fasciolosis. They also improve diagnostic sensitivity and specificity by establishing a robust methodological framework for biomarker discovery, as well as for subsequent functional analyses and large-scale evaluations.