Gutala Ramana V, Reddy P Hemachandra
Neurogenetics Laboratory, Neurological Sciences Institute, Oregon Health & Science University, Beaverton, OR 97006, USA.
J Neurosci Methods. 2004 Jan 15;132(1):101-7. doi: 10.1016/j.jneumeth.2003.09.005.
The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.
对患有神经退行性疾病的大脑中的基因表达进行测量是脑研究的一个主要领域。我们研究的目的是确定定量实时PCR是否能够测量死后间隔超过12小时的大脑中的信使核糖核酸(mRNA)表达。在本文中,我们检测了来自阿尔茨海默病(AD)患者(n = 13)和非痴呆正常对照受试者(n = 6)脑标本的RNA质量。为了确定AD中一个不受调控的内源性参考基因,我们测量了常用参考基因β-肌动蛋白、18S核糖体RNA和甘油醛-3-磷酸脱氢酶(GAPDH)的mRNA表达。此外,我们还确定了死后间隔、脑重量或死亡年龄是否会影响mRNA表达。我们的实时PCR分析结果表明,在所有脑标本中都能检测到β-肌动蛋白、18S核糖体RNA、GAPDH以及突触素(一种已知的AD标志物)的mRNA表达。此外,通过实时PCR分析,我们发现β-肌动蛋白和18S核糖体RNA在AD和对照受试者的脑标本中表达存在差异,而GAPDH在AD和对照脑标本中的表达相似。这些发现表明,GAPDH可作为AD脑研究中的内源性参考基因。一项比较基因表达分析还表明,与对照脑标本相比,AD脑标本中的突触素表达下调。