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

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is widely used to accurately assess target gene expression. Evaluating gene expression requires the selection of appropriate reference genes. To identify reliable reference genes for Toxoplasma gondii (T. gondii) under varying concentrations of broxaldine (BRO), we employed the ΔCt method, BestKeeper, NormFinder, GeNorm, and the comprehensive web-based platform RefFinder to assess the expression stability of ten candidate reference genes in T. gondii. Herein, our findings reveal that the stability of these candidate reference genes is influenced by different experimental conditions. Under normal conditions, the most stable genes were TGME49_205470 and TGME49_226020. However, the most stable genes differed when BRO concentrations were at 1, 2, and 4 μg/mL. Across all samples, TGME49_247220 and TGME49_235930 were identified as the most stable reference genes. Moreover, we also confirmed the stability of TGME49_247220 and TGME49_235930 as reference genes through RT-qPCR assays. The present study provides a foundation for applying the RT-qPCR method to investigate target gene expression following BRO treatment in T. gondii.