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Abstract <p>Real-time quantitative PCR (RT-qPCR) is a widely used method for analyzing cellular responses. However, reliable results depend on the selection of appropriate reference genes, for which data are scarce in studies using hamsters as an animal model. In this study, we employed the R package RefSeeker, which integrates NormFinder, geNorm, BestKeeper, delta-Ct, and RefFinder algorithms, to identify the most stably expressed candidate reference genes in kidney, liver, and lung samples of golden Syrian hamsters. The animals were subjected to vaccination with aluminum hydroxide adjuvant, <i>Leptospira</i> bacterin, vaccine vectors (<i>Mycobacterium bovis</i> BCG and attenuated <i>Salmonella</i> Typhimurium), or infection with <i>Leptospira interrogans</i>. The most stable gene identified in the overall analysis (including all samples and conditions), as well as under vaccination with aluminum hydroxide, <i>Salmonella</i> Typhimurium, and control (PBS), was <i>ppib</i>. Under bacterin and <i>M. bovis</i> BCG conditions, <i>rps29</i> was the most suitable reference gene, while <i>ppia</i> showed the highest stability during infection. These findings were validated using the <i>ifn</i><Emphasis Type="BoldItalic">-</Emphasis><b>γ</b> gene as a target, with expression levels assessed under the BCG vaccination condition. Our results demonstrate that sample type and experimental condition significantly influence endogenous gene expression. Therefore, the use of universal reference genes is not recommended, and gene selection should be carefully tailored for each experiment. This work provides a validated framework for accurate normalization of gene expression in preclinical models using hamsters.</p>

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Identification of Stable Reference Genes for RT-qPCR in Golden Syrian Hamster under Vaccination and Infection Conditions

  • 期刊论文

  • 发表日期：

    2025年11月17日

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  • T. T. Bunde,
  • A. C. K. Pedra,
  • M. A. C. Maia,
  • N. R. de Oliveira,
  • O. A. Dellagostin,
  • T. L. O. Bohn

摘要

Abstract

Real-time quantitative PCR (RT-qPCR) is a widely used method for analyzing cellular responses. However, reliable results depend on the selection of appropriate reference genes, for which data are scarce in studies using hamsters as an animal model. In this study, we employed the R package RefSeeker, which integrates NormFinder, geNorm, BestKeeper, delta-Ct, and RefFinder algorithms, to identify the most stably expressed candidate reference genes in kidney, liver, and lung samples of golden Syrian hamsters. The animals were subjected to vaccination with aluminum hydroxide adjuvant, Leptospira bacterin, vaccine vectors (Mycobacterium bovis BCG and attenuated Salmonella Typhimurium), or infection with Leptospira interrogans. The most stable gene identified in the overall analysis (including all samples and conditions), as well as under vaccination with aluminum hydroxide, Salmonella Typhimurium, and control (PBS), was ppib. Under bacterin and M. bovis BCG conditions, rps29 was the most suitable reference gene, while ppia showed the highest stability during infection. These findings were validated using the ifn-γ gene as a target, with expression levels assessed under the BCG vaccination condition. Our results demonstrate that sample type and experimental condition significantly influence endogenous gene expression. Therefore, the use of universal reference genes is not recommended, and gene selection should be carefully tailored for each experiment. This work provides a validated framework for accurate normalization of gene expression in preclinical models using hamsters.