The quantification of gene expression in an animal model of brain ischaemia using TaqMan™ real-time RT-PCR
The quantification of gene expression in an animal model of brain ischaemia using TaqMan™ real-time RT-PCR
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DOI：
10.1016/s0169-328x(02)00417-5
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发表时间：
2002-10-15
期刊：
影响因子：
--
通讯作者：
Preece, P
中科院分区：
文献类型：
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作者：
Bond, BC;Virley, DJ;Preece, P
mRNA的表达水平通常测量为测试基因与参考基因的比率。假设参考基因如P-肌动蛋白或亲环蛋白不受治疗影响，并作为稳态对照。TaqMan（TM）实时RT-PCR用于在脑缺血（tMCAO）的大鼠模型中测试这些假设。在测量24个基因后，我们发现该动物模型中的参考基因不符合稳态对照的标准。神经元丢失、神经胶质增生和白细胞流入病变脑导致细胞群的重大紊乱。与测试基因一样，参考基因的mRNA反映了这些变化。特定的mRNA水平根据它们被标准化的参考基因的选择而变化。在解决参考基因问题的过程中，发现缺血影响的大鼠大脑半球中白血病抑制因子、巢蛋白和甘丙肽的mRNA增加。结果报告了另外21个基因和数学和统计方法进行了描述，允许在这项研究中分数倍的mRNA变化进行检测。(C)2002 Elsevier Science B. V.保留所有权利。
Expression levels of mRNA are commonly measured as a ratio of test to reference gene. The assumption is that reference genes such as P-actin or cyclophilin are unaffected by treatment and act as steady-state controls. TaqMan(TM) real-time RT-PCR was used to test these assumptions in a rat model of cerebral ischaemia (tMCAO). Following measurement of 24 genes, we show that reference genes in this animal model fail the criteria for steady-state controls. Neuronal loss, glial proliferation and an influx of leukocytes into the lesioned brain result in major disturbance to cell populations. The mRNA for reference genes, as for test genes, reflects these changes. Specific mRNA levels vary according to the choice of reference gene to which they are normalised. In the process of resolving reference gene issues, mRNA increases were discovered for leukaemia inhibitory factor, nestin and galanin in rat brain hemispheres affected by ischaemia. Results are reported for a further 21 genes and mathematical and statistical methods are described that allow in this study fraction-fold changes in mRNA to be detected. (C) 2002 Elsevier Science B.V. All rights reserved.