Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue
Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue
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DOI：
10.1016/j.jalz.2009.11.002
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发表时间：
2010-11-01
影响因子：
14
通讯作者：
Dodd, Peter R.
中科院分区：
文献类型：
--
作者：
Gebhardt, Florian M.;Scott, Heather A.;Dodd, Peter R.
背景定量真实的时间反转录聚合酶链反应（qRT PCR）是研究mRNA表达的常用技术（管家）被广泛用于校正阿尔茨海默病（AD）的装载和RNA质量差异方法采用qRT PCR方法对8个推测的管家基因进行了定量分析，在15个对照组的四个脑区中，结果12例AD和10例AD/Lewy body disease（LBD）患者的RNA质量低于对照组，常用的管家因子如甘油醛-3磷酸脱氢酶（GAPDH）和13肌动蛋白在AD和AD/LBD患者中的总体表达较低。LBD病例中RPL 13和18 S比对照中表达的管家蛋白最稳定。测试突触素和半乳糖醛酸酸性蛋白用于评估标准化定量。通过使用不同的管家蛋白，我们证实，AD患者突触素表达下调，而胶质细胞酸性蛋白表达增加。结论在所有测试的候选人中，RPL 13是对照组和AD患者尸检脑组织样品qRT PCR研究的最佳管家。RNA质量也应该评估，并根据该指标标准化数据。
Background Quantitative real time reverse transcription polymerase chain reaction (qRT PCR) is a popular technique for mRNA expression studies Normalization to an endogenous reference tran script (housekeeper) is widely used to correct for differences in loading and RNA quality Alzheimer's disease (AD) alters brain metabolism The stability of housekeeper transcript expression must be care fully validatedMethods qRT PCR was used to assess eight putative housekeeper transcripts in four brain regions from 15 control, 12 AD and 10 AD/Lewy body disease (LBD) casesResults RNA quality is lower in AD and AD/LBD than in controls Frequently used housekeepers such as glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and 13 actin had lower overall expression in AD and AD/LBD cases than in controls RPL13 and 18S were the most stably expressed house keepers tested Synaptophysin and ghat fibrillary acidic protein were used to evaluate normalized quantification By using different housekeepers we confirmed that synaptophysin expression was down-regulated in AD cases, whereas glial fibrillary acidic protein expression was increasedConclusions Among all candidates tested, RPL13 was the best housekeeper for qRT PCR studies in autopsy brain tissue samples from controls and AD cases RNA quality should be assessed and data normalized on this index as well (C) 2010 The Alzheimer's Association All rights reserved