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Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart
Daniel Benak1, Dita Sotakova-Kasparova1, Jan Neckar1
1Department of Developmental Cardiology, Institute of Physiology of the Czech Academy of Sciences, Prague, 142 20, Czech Republic.
Molecular and Cellular Biochemistry
|July 14, 2019
Summary
Selecting appropriate reference genes is crucial for accurate gene expression analysis in chronically hypoxic rat hearts. This study identifies the best reference genes for the left and right ventricles under varying hypoxia conditions.
Area of Science:
- Cardiovascular Physiology
- Molecular Biology
- Gene Expression Analysis
Background:
- Chronic hypoxia enhances cardiac tolerance to ischemia/reperfusion injury.
- Accurate gene expression studies require stable reference genes for normalization.
- No validated reference genes exist for the myocardium in chronically hypoxic rat models.
Purpose of the Study:
- To identify and validate the most stable reference genes in the left (LV) and right (RV) ventricles of rats adapted to chronic normobaric hypoxia (CNH).
- To compare reference gene stability between normoxic and chronically hypoxic conditions, and between LV and RV.
- To provide essential tools for reliable gene expression analysis in cardiac research under hypoxia.
Main Methods:
- Establishment of a rat model for chronic normobaric hypoxia (12% or 10% O2 for 3 weeks).
- Quantitative real-time PCR (RT-qPCR) to assess expression levels of candidate reference genes.
- Utilizing NormFinder, geNorm, and BestKeeper algorithms to evaluate gene stability.
Main Results:
- Identified Top1, Nupl2, Rplp1, Ywhaz, Hprt1 as top reference genes for LV under milder CNH.
- Identified Top1, Ywhaz, Sdha, Nupl2, Tomm22 as top reference genes for LV under stronger CNH.
- Identified Hprt1, Nupl2, Gapdh, Top1, Rplp1 for RV under milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for RV under stronger CNH.
Conclusions:
- Validated specific reference genes for LV and RV in CNH rat models.
- Demonstrated that optimal reference genes differ between ventricles (LV vs. RV).
- Highlighted the dependence of reference gene selection on the experimental hypoxia protocol intensity.
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