Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart
Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart
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DOI：
10.1007/s11010-019-03584-x
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发表时间：
2019-11-01
影响因子：
4.3
通讯作者：
Hlavackova, Marketa
中科院分区：
文献类型：
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作者：
Benak, Daniel;Sotakova-Kasparova, Dita;Hlavackova, Marketa
对慢性低氧的适应使心脏对缺血/再灌注损伤有更强的耐受性。为了用RT-qPCR评价慢性低氧适应后基因表达的变化，选择合适的参考基因是非常必要的。在一个慢性缺氧的大鼠模型中，在心肌中还没有发现特定的参考基因。本研究旨在筛选慢性低氧和常氧大鼠左(LV)和右(RV)的最佳参考基因。SD大鼠适应持续常压低氧(CNH；12%O-2或10%O-2)3周。用RT-qPCR检测候选基因的表达水平。用NormFinder、geNorm和BestKeeper算法评价基因的稳定性。LV的5个参考基因中，Top1、Nupl2、Rplp1、Ywhaz、Hprt1代表轻度CNH，Top1、Ywhaz、Sdha、Nupl2、Tomm22代表CNH强度。在RV中，轻度CNH的前5位基因是Hprt1、Nupl2、GAPDH、Top1、Rplp1，而重度CNH的前5位基因是Tomm22、GAPDH、Hprt1、Nupl2、Top1。本研究提供了CNH大鼠左、右室参考基因的验证，并表明两个脑室中合适的参考基因不同，并且依赖于实验方案。
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% O-2 or 10% O-2) 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.