Evaluation of reference genes for gene expression studies in mouse and N2a cell ischemic stroke models using quantitative real-time PCR.
Evaluation of reference genes for gene expression studies in mouse and N2a cell ischemic stroke models using quantitative real-time PCR.
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DOI：
10.1186/s12868-018-0403-6
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发表时间：
2018-02-01
期刊：
影响因子：
2.4
通讯作者：
Lu B
中科院分区：
文献类型：
--
作者：
Kang Y;Wu Z;Cai D;Lu B
实时逆转录定量聚合酶链反应（RT-qPCR）是评估基因转录mRNA水平的关键工具。可靠的RT-qPCR结果很大程度上取决于对合适参考基因的标准化。大脑中动脉闭塞（MCAO）和氧-糖剥夺/复氧（OGD/R）是研究缺血性卒中的常用模型。然而，在这两种模型中用于RNA分析的合适参考基因尚未确定。在这项研究中，我们评估了六个候选管家基因的表达水平，并选择了最合适的参考基因用于MCAO小鼠和OGD/R损伤的N2 a细胞的皮质的RT-qPCR分析。使用四个软件程序geNorm、Normandy、BestKeeper和Referral来验证候选参考基因的稳定性。结果显示，HPRT和18 S是MCAO小鼠皮质中最稳定的参考基因，β-肌动蛋白和亲环蛋白是OGD/R损伤的N2 a细胞中最稳定的参考基因;相比之下，GAPDH和Sdha分别是MCAO小鼠皮质和OGD/R损伤的N2 a细胞中最不稳定的基因。此外，HPRT、18 S和亲环蛋白的组合最适合于在MCAO小鼠的皮质分析中进行标准化，并且β-肌动蛋白、亲环蛋白、GAPDH和18 S的组合最适合于OGD/R损伤的N2 a细胞的分析。本研究为进一步的体内和体外缺血性卒中的RT-qPCR分析提供了适当的参考基因，并证明了在可变条件下验证参考基因用于RNA分析的必要性。本文的在线版本（10.1186/s12868-018-0403-6）包含补充材料，可供授权用户使用。
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen–glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined. In this study, we evaluated the expression levels of six candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR analyses of the cortices of MCAO mice and OGD/R-injured N2a cells. Four software programs, geNorm, NormFinder, BestKeeper and RefFinder, were used to validate the stabilities of the candidate reference genes. The results revealed that HPRT and 18S were the most stable reference genes in the cortices of MCAO mice and that β-actin and cyclophilin were the most stable reference genes in the OGD/R-injured N2a cells; in contrast, GAPDH and Sdha were the least stable genes in the cortices of MCAO mice and the OGD/R-injured N2a cells, respectively. Moreover, a combination of HPRT, 18S and cyclophilin was most suitable for normalization in analyses of the cortices of MCAO mice, and a combination of β-actin, cyclophilin, GAPDH, and 18S was most suitable for analyses of the OGD/R-injured N2a cells. This study provides appropriate reference genes for further RT-qPCR analyses of in vivo and in vitro ischemic stroke and demonstrates the necessity of validating reference genes for RNA analyses under variable conditions. The online version of this article (10.1186/s12868-018-0403-6) contains supplementary material, which is available to authorized users.
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