Hoerndli Frédéric J, Toigo Marco, Schild Andreas, Götz Jürgen, Day Philip J
Division of Psychiatry Research, University of Zürich, August Forel Strasse 1, 8008 Zürich, Switzerland.
Anal Biochem. 2004 Dec 1;335(1):30-41. doi: 10.1016/j.ab.2004.08.028.
Transcriptomic methods are widely used as an initial approach to gain a mechanistic insight into physiological and pathological processes. Because differences in gene regulation to be assessed by RNA screening methods (e.g., SAGE, Affymetrix GeneChips) can be very subtle, these techniques require stable reference genes for accurate normalization. It is widely known that housekeeping genes, which are routinely used for normalization, can vary significantly depending on the tissue, and experimental test. In this study, we aimed at identifying stable reference genes for a fibrillar Abeta(42) peptide-treated, human tau-expressing SH-SY5Y neuroblastoma cell line derived to model aspects of Alzheimer's disease in tissue culture. We selected genes exhibiting potential normalization characteristics from public databases to create a custom-made microarray allowing the identification of reference genes for low, intermediate, and abundant mRNAs. A subset of these candidates was subjected to quantitative real-time polymerase chain reaction and was analyzed with geNorm software. By doing so, we were able to identify GAPD, M-RIP, and POLR2F as stable and usable reference genes irrespective of differentiation status and Abeta(42) treatment.
转录组学方法被广泛用作初步手段，以深入了解生理和病理过程的机制。由于通过RNA筛选方法（如SAGE、Affymetrix基因芯片）评估的基因调控差异可能非常细微，这些技术需要稳定的参考基因进行准确的标准化。众所周知，通常用于标准化的管家基因会因组织和实验测试的不同而有显著差异。在本研究中，我们旨在为经纤维状β淀粉样蛋白（Aβ）42肽处理、表达人tau蛋白的SH-SY5Y神经母细胞瘤细胞系鉴定稳定的参考基因，该细胞系用于在组织培养中模拟阿尔茨海默病的某些方面。我们从公共数据库中选择具有潜在标准化特征的基因，制作定制微阵列，以鉴定低丰度、中等丰度和高丰度mRNA的参考基因。对这些候选基因中的一部分进行定量实时聚合酶链反应，并使用geNorm软件进行分析。通过这样做，我们能够鉴定出甘油醛-3-磷酸脱氢酶（GAPD）、膜相关的富含脯氨酸的蛋白质（M-RIP）和RNA聚合酶Ⅱ亚基F（POLR2F）作为稳定且可用的参考基因，无论细胞分化状态和Aβ42处理情况如何。
