Impact of Reference Gene Choice on Accurate Gene Expression Analysis Under Emerging Contaminant Exposure

Accurate interpretation of gene expression in organisms exposed to emerging contaminants (ECs) requires reliable normalization using stable reference genes. In this study, six candidate housekeeping genes (EEF1A1, GAPDH, ACTB, TBP, RAG1, and PKM) were evaluated in fish (Labeo catla) liver under exposure to chlorpyrifos, triclosan, and oxytetracycline. Expression stability was assessed using geNorm, NormFinder, BestKeeper, and the ΔC_t method, with RefFinder providing an integrated ranking. EEF1A1 consistently showed the highest stability, whereas PKM and RAG1 were the least reliable. Normalization of the xenobiotic response gene CYP1A1 with stable (EEF1A1) versus unstable (PKM, RAG1) genes revealed that inappropriate reference gene choice produced misleading expression profiles. These results highlight the critical importance of empirical reference gene validation for accurate qRT-PCR-based gene expression analysis, particularly in ecotoxicological studies involving exposure to complex mixtures of contaminants.