Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease
Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease
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DOI：
10.1016/j.biopha.2017.01.091
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发表时间：
2017-04-01
影响因子：
7.5
通讯作者：
Majerowicz, David
中科院分区：
文献类型：
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作者：
Almeida-Oliveira, Fernanda;Leandro, Joao G. B.;Majerowicz, David
肥胖和糖尿病都是代谢性疾病，发病率正在上升。与这些疾病相关的基因表达动力学是识别参与相关生物学过程的基因的基础。qPCR是一种灵敏的mRNA定量技术，也是基因表达研究中最常用的方法。然而，这些结果的可靠性直接受到数据规范化的影响。由于内参基因是主要的归一化方法，本工作旨在确定1型糖尿病或肥胖小鼠脂肪组织中qPCR的内参基因。我们选取了12个常用的内参基因。在三种不同的实验方案下，分析了这些基因在小鼠脂肪组织中的表达：1)未经治疗的动物；2)高脂肪动物；3)链脲佐菌素治疗动物。使用四种不同的算法分析基因表达稳定性。我们的数据表明，tata结合蛋白在对照动物的脂肪组织中稳定表达。该基因在分析正常小鼠和肥胖小鼠的棕色脂肪组织时也有参考价值。线粒体ATP合成酶F1复合体基因在正常小鼠和肥胖小鼠皮下和趾周脂肪组织中稳定表达。此外，该基因是链脲佐菌素处理动物脂肪组织qPCR正常化的最佳参考。这些结果表明，在所有的实验条件下，不存在完全稳定的基因。综上所述，选择合适的基因是保证qPCR可靠性的前提，必须针对不同的实验方案分别进行。(C) 2017 Elsevier Masson SAS。版权所有。
Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols. (C) 2017 Elsevier Masson SAS. All rights reserved.