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

被引：0
作者：
Daniel Benak
Dita Sotakova-Kasparova
Jan Neckar
Frantisek Kolar
Marketa Hlavackova
机构：
[1] Institute of Physiology of the Czech Academy of Sciences,Department of Developmental Cardiology
来源：
Molecular and Cellular Biochemistry | 2019年 / 461卷
关键词：
Reference genes; RT-qPCR; Heart; Left ventricle; Chronic hypoxia; Rat;
D O I：
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学科分类号：
摘要：
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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页码：15 / 22
页数：7
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[1]  
Neckar J(2004)Myocardial infarct size-limiting effect of chronic hypoxia persists for five weeks of normoxic recovery Physiol Res 53 621-628
[2]  
Ostadal B(2004)Molecular mechanisms of cardiac protection by adaptation to chronic hypoxia Physiol Res 53 S3-13
[3]  
Kolar F(2007)Cardiac adaptation to chronic high-altitude hypoxia: beneficial and adverse effects Respir Physiol Neurobiol 158 224-236
[4]  
Kolar F(2016)Analysis of the stability of housekeeping gene expression in the left cardiac ventricle of rats submitted to chronic intermittent hypoxia J Bras Pneumol 42 211-214
[5]  
Ostadal B(2008)Analyzing real-time PCR data by the comparative C(T) method Nat Protoc 3 1101-1108
[6]  
Ostadal B(2009)The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments Clin Chem 55 611-622
[7]  
Kolar F(2002)Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol 3 research0034.1-0034.11
[8]  
Julian GS(2012)Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression BMC Res Notes 5 124-1043
[9]  
Oliveira RW(2010)‘Desperate house genes’: the dramatic example of hypoxia Br J Cancer 102 1037-1366
[10]  
Tufik S(2016)Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiment Naunyn Schmiedebergs Arch Pharmacol 389 1353-748
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