Reference gene analysis and its use for kinase expression profiling in Fasciola hepatica

Area of Science:

• Parasitology
• Molecular Biology
• Zoonotic Diseases

Background:

• Fasciola hepatica causes fasciolosis, a significant foodborne zoonosis in humans and livestock.
• Accurate gene expression quantification across parasite life stages is vital for identifying anthelmintic drug targets.

Purpose of the Study:

• To identify and validate stable reference genes for quantitative real-time PCR (qRT-PCR) in Fasciola hepatica research.
• To establish reliable gene expression normalization for studies on parasite development and drug targeting.

Main Methods:

• Evaluated expression stability of eight candidate reference genes using NormFinder, geNorm, BestKeeper, and comparative ΔCT methods.
• Compared gene expression across intra-mammalian life stages and in vitro-cultured juveniles.
• Quantified expression of five target kinases (Abl1, Abl2, PKC, Akt1, Plk1) using validated reference genes.

Main Results:

• Glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd) were the most stable genes for intra-mammalian stages.
• Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7) were optimal for in vitro-cultured juveniles.
• The commonly used housekeeping gene gapdh showed differential expression and is not recommended.
• Distinct expression patterns for five kinases were revealed, suggesting important biological functions.

Conclusions:

• Validated reference genes (Fheprs, Fhtbcd, Fhpsmb7) provide a more accurate basis for Fasciola hepatica gene expression analysis.
• These validated genes should replace gapdh in future studies for improved reliability in drug target identification and developmental research.
• The findings facilitate deeper understanding of Fasciola hepatica biology and the development of novel anthelmintic strategies.