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Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue.
BMC Molecular Biology ( IF 4.619 ) Pub Date : 2008-05-06 , DOI: 10.1186/1471-2199-9-46
David T R Coulson 1 , Simon Brockbank , Joseph G Quinn , Suzanne Murphy , Rivka Ravid , G Brent Irvine , Janet A Johnston
Affiliation  

BACKGROUND Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. RESULTS The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan(R) Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. CONCLUSION This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

中文翻译:

鉴定用于人脑组织中 RT qPCR 基因表达数据标准化的有效参考基因。

背景 死后人脑中基因表达的研究有助于了解神经退行性疾病的病理生理学,包括阿尔茨海默病 (AD)、帕金森病 (PD) 和路易体痴呆 (DLB)。定量实时 PCR (RT qPCR) 通常用于分析基因表达。使用 RT qPCR 获得的结果的有效性取决于准确的数据标准化。参考基因通常用于标准化 RT qPCR 数据。鉴于某些常用参考基因​​的表达在某些条件下会发生改变,本研究旨在确定哪些参考基因在 AD、PD 或 DLB 患者的死后脑组织中稳定表达。结果 本研究调查了 8 个候选参考基因(泛素 C [UBC]、酪氨酸-3-单加氧酶 [YWHAZ]、RNA 聚合酶 II 多肽 [RP II]、羟甲基胆碱合酶 [HMBS]、TATA 盒结合蛋白 [TBP]、β-2-微球蛋白 [B2M]、3-磷酸甘油醛脱氢酶 [GAPDH]和琥珀酸脱氢酶复合物-亚基 A,[SDHA]) 在 6 名 AD、6 名 PD、6 名 DLB 受试者的小脑和内侧颞回,以及使用 RT qPCR(TaqMan(R) 基因表达分析)的 5 个匹配对照。使用geNorm分析基因表达稳定性,以在每个疾病组中按照稳定性递减的顺序对候选基因进行排序。推荐用于每种疾病状态下准确数据标准化的最佳基因数量是通过成对变异分析确定的。
更新日期:2019-11-01
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