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Systematic analysis of candidate reference genes for gene expression analysis in hyperoxia-based mouse models of
Mary Linge1, Marius A Möbius1, Angela Rösen-Wolff1
1Department of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.
Insights
This study identifies the most stable reference genes, Hprt, Tbp, and Hmbs, for analyzing gene expression in mouse models of bronchopulmonary dysplasia. These findings improve the accuracy of gene expression analysis in hyperoxia-induced lung injury research.
Area of Science:
- Pulmonary Medicine
- Molecular Biology
- Genetics
Background:
- Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting preterm infants.
- Mouse models of hyperoxia-induced lung injury are crucial for studying BPD pathogenesis and therapeutics.
- Accurate gene expression analysis in these models relies on validated reference genes for RT-qPCR.
Purpose of the Study:
- To systematically analyze and identify stable reference genes for gene expression studies in neonatal mouse lungs under hyperoxia.
- To establish reliable normalization factors for RT-qPCR in hyperoxia-induced lung injury models.
- To validate the identified reference genes and normalization factors using a relevant gene target (Il6).
Main Methods:
- Systematic analysis of 12 potential reference genes in neonatal mouse lung tissue exposed to room air or hyperoxia, plus adult controls.
- Utilized published software algorithms to assess gene expression stability.
- Validated findings by comparing Il6 gene and protein expression using the identified reference genes/factors.
Main Results:
- Identified Hprt, Tbp, and Hmbs as the most stable reference genes.
- Proposed Hprt/Sdha and Hprt/Rpl13a combinations as effective normalization factors.
- Demonstrated the utility of these reference genes/factors in validating Il6 expression.
Conclusions:
- Hprt, Tbp, and Hmbs are recommended as stable reference genes for hyperoxia-induced neonatal lung injury mouse models.
- The proposed normalization factors (Hprt/Sdha or Hprt/Rpl13a) can enhance the accuracy of gene expression analysis.
- These validated reference genes will facilitate more reliable research into BPD pathogenesis and treatment.
Abstract:
Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants. Mouse models of hyperoxia-induced lung injury are often used to study pathogenesis and potential therapeutic approaches of BPD. Beside histological studies, gene expression analysis of lung tissue is typically used as experimental readout. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the standard method for gene expression analysis; however, the accuracy of the quantitative data depends on the appropriate selection of reference genes. No data on validated reference genes for hyperoxia-induced neonatal lung injury in mice are available. In this study, 12 potential reference genes were systematically analyzed for their expression stability in lung tissue of neonatal mice exposed to room air or hyperoxia and healthy adult controls using published software algorithms. Analysis of gene expression data identified Hprt, Tbp, and Hmbs as the most stable reference genes and proposed combinations of Hprt/Sdha or Hprt/Rpl13a as potential normalization factors. These reference genes and normalization factors were validated by comparing Il6 gene and protein expression and may facilitate accurate gene expression analysis in lung tissues of similar designed studies.
