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Updated: Jun 29, 2026
Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes
Published on: August 9, 2011
Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection
Lethicia Souza Tavares1, Roberta Lane Oliveira-Silva2, Marcelo Tigre Moura3
1Departamento de Biologia, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
Abstract:
RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with Listeria monocytogenes. The pMØ were infected in vitro with L. monocytogenes or vehicle for 4 h. Mice were injected with L. monocytogenes (or vehicle) and euthanized 24 h post-injection. The RGs came from a multispecies primer set, from the literature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CT and RefFinder. B2m-H3f3a-Ppia were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. Actβ-Ubc-Ppia were the best RGs for spleen but modestly impacted the cytokine relative expression. Hence, mouse models of L. monocytogenes require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.
Insights
Finding the right reference genes (RGs) is crucial for accurate RT-qPCR analysis in mouse models. This study identified specific RGs for macrophages and spleen infected with Listeria monocytogenes, improving gene expression profiling.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Quantitative real-time PCR (RT-qPCR) is essential for analyzing gene expression.
- Accurate normalization in RT-qPCR relies on stable reference genes (RGs).
- Identifying context-specific RGs is critical for reliable results in biological studies.
Purpose of the Study:
- To identify and validate stable reference genes for RT-qPCR in mouse macrophages (pMØ) and spleen.
- To evaluate the impact of different RGs on gene expression analysis during Listeria monocytogenes infection.
- To ensure accurate gene expression profiling in mouse models of infection.
Main Methods:
- In vitro infection of mouse peritoneal macrophages (pMØ) with L. monocytogenes.
- In vivo infection of mice with L. monocytogenes, followed by spleen collection.
- Selection of candidate RGs from various sources (multispecies primers, literature, de novo design).
- Statistical analysis of RG stability using GeNorm, NormFinder, BestKeeper, Delta-CT, and RefFinder algorithms.
Main Results:
- For infected pMØ, B2m, H3f3a, and Ppia were identified as the most stable RGs.
- For infected spleen tissue, Actb, Ubc, and Ppia emerged as the best RGs.
- The choice of RGs influenced the estimation of cytokine relative expression in both models.
- Context-specific RGs optimized gene expression profiling in L. monocytogenes-infected mouse models.
Conclusions:
- Mouse models of L. monocytogenes infection necessitate the use of context-specific reference genes for RT-qPCR.
- The identified RGs (B2m-H3f3a-Ppia for pMØ; Actb-Ubc-Ppia for spleen) enhance the accuracy of gene expression analysis.
- This study underscores the importance of rigorous RG selection for reliable molecular profiling in infectious disease research.
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