Reference Gene Selection for RT-qPCR Normalization in Toxoplasma gondii Exposed to Broxaldine

Area of Science:

• Molecular Biology
• Parasitology
• Bioinformatics

Background:

• Accurate gene expression analysis using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) relies on stable reference genes.
• Toxoplasma gondii (T. gondii) research requires validated reference genes for reliable gene expression studies, especially under drug treatment conditions.

Purpose of the Study:

• To identify the most stable reference genes for RT-qPCR analysis in T. gondii exposed to varying concentrations of broxaldine (BRO).
• To provide validated reference genes for future studies investigating the effects of broxaldine on T. gondii gene expression.

Main Methods:

• Evaluation of ten candidate reference genes using multiple statistical algorithms: ΔCt method, BestKeeper, NormFinder, and GeNorm.
• Comprehensive analysis of gene expression stability using the RefFinder web-based platform.
• Validation of selected reference genes through RT-qPCR assays.

Main Results:

• Reference gene stability in T. gondii is significantly affected by broxaldine treatment.
• Under normal conditions, TGME49_205470 and TGME49_226020 were the most stable genes.
• Across all experimental conditions (including varying BRO concentrations), TGME49_247220 and TGME49_235930 demonstrated the highest expression stability.

Conclusions:

• TGME49_247220 and TGME49_235930 are validated as the most reliable reference genes for RT-qPCR in T. gondii under broxaldine treatment.
• The findings establish a foundation for accurate gene expression profiling in T. gondii following broxaldine exposure.
• This research is critical for understanding drug mechanisms and resistance in T. gondii infections.