Penna Ilaria, Vella Serena, Gigoni Arianna, Russo Claudio, Cancedda Ranieri, Pagano Aldo
Oncology, Biology, and Genetics Department (DOBiG), University of Genoa, Largo R. Benzi, 10, 16132 Genoa, Italy; E-Mails:
Int J Mol Sci. 2011;12(9):5461-70. doi: 10.3390/ijms12095461. Epub 2011 Aug 26.
The most frequently used technique to study the expression profile of genes involved in common neurological disorders is quantitative real-time RT-PCR, which allows the indirect detection of very low amounts of selected mRNAs in tissue samples. Expression analysis by RT-qPCR requires an appropriate normalization to the expression level of genes characterized by a stable, constitutive transcription. However, the identification of a gene transcribed at a very stable level is difficult if not impossible, since significant fluctuations of the level of mRNA synthesis often accompanies changes of cell behavior. The aim of this study is to identify the most stable genes in postmortem human brain samples of patients affected by Alzheimer's disease (AD) suitable as reference genes. The experiments analyzed 12 commonly used reference genes in brain samples from eight individuals with AD and seven controls. After a careful analysis of the results calculated by geNorm and NormFinder algorithms, we found that CYC1 and EIF4A2 are the best reference genes. We remark on the importance of the determination of the best reference genes for each sample to be analyzed and suggest a practical combination of reference genes to be used in the analysis of human postmortem samples.
研究常见神经疾病相关基因表达谱最常用的技术是定量实时逆转录聚合酶链反应（RT-PCR），它能够间接检测组织样本中极少量的特定信使核糖核酸（mRNA）。通过RT-qPCR进行表达分析需要对以稳定的组成型转录为特征的基因表达水平进行适当的标准化。然而，要鉴定转录水平非常稳定的基因即便不是不可能也是很困难的，因为mRNA合成水平的显著波动常常伴随着细胞行为的变化。本研究的目的是在阿尔茨海默病（AD）患者的死后人类脑样本中鉴定出最稳定的基因作为参考基因。实验分析了来自8名AD患者和7名对照的脑样本中的12个常用参考基因。在仔细分析由geNorm和NormFinder算法计算得出的结果后，我们发现CYC1和EIF4A2是最佳参考基因。我们强调了为每个待分析样本确定最佳参考基因的重要性，并建议了一种用于人类死后样本分析的参考基因实用组合。
