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Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart

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Abstract

Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague–Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3 weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.

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Acknowledgements

This research was funded by CHARLES UNIVERSITY GRANT AGENCY, Grant Number 200317 and GRANT AGENCY OF THE CZECH REPUBLIC, Grant Numbers 16-12420Y and 19-04790Y. We kindly thank Dr. Matus Sotak for help with manuscript editing.

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Authors and Affiliations

  1. Department of Developmental Cardiology, Institute of Physiology of the Czech Academy of Sciences, Prague, 142 20, Czech Republic

    Daniel Benak, Dita Sotakova-Kasparova, Jan Neckar, Frantisek Kolar & Marketa Hlavackova

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  1. Daniel Benak
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  2. Dita Sotakova-Kasparova
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  3. Jan Neckar
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  4. Frantisek Kolar
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Correspondence to Marketa Hlavackova.

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The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All procedures have been performed in accordance with the ethical standards and with the approvement of the Ethical Committee of the Institute of Physiology CAS in Prague.

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Benak, D., Sotakova-Kasparova, D., Neckar, J. et al. Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart. Mol Cell Biochem 461, 15–22 (2019). https://doi.org/10.1007/s11010-019-03584-x

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