Identification and validation of stable reference genes for RT-qPCR analysis in blood samples from golden Syrian hamsters under different immunological conditions

BACKGROUND: The golden Syrian hamster is the main experimental model for leptospirosis. However, there are no validated reference genes for reverse transcription quantitative PCR (RT-qPCR) analysis of blood samples. METHODS AND RESULTS: The expression stability of 11 candidate reference genes was evaluated in hamster blood under distinct immunological conditions, including Leptospira interrogans infection and different immunization strategies. Stability was assessed using BestKeeper, geNorm, NormFinder, and the comparative ΔCt method, with the consensus ranking calculated using the RefFinder approach implemented in the RefSeeker R package. The most stable reference gene was ppia, followed by gapdh and ppib, whereas actb was the least stable gene in the ranking. Normalization of tnf-α expression using actb resulted in an approximately 10-fold overestimation compared with ppia, demonstrating the impact of reference gene choice on data interpretation. CONCLUSIONS: These findings support ppia as a reliable reference gene for RT-qPCR normalization in hamster blood under both infection and vaccination conditions.