Abstract
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.
Highlights
Alzheimer’s disease (AD) is a progressive neurodegenerative pathology affecting hippocampus, temporal and/or frontal cerebral cortex lobes
In this context the most frequently used approach to study the expression profile of genes involved in AD is quantitative real-time RT-PCR (RT-qPCR), which allows the indirect detection of very low amounts of selected messenger RNAs in tissue samples
In order to compare expression levels of target genes in different tissues at the same time, it is crucial to normalize all samples by the same set of reference genes
Summary
Alzheimer’s disease (AD) is a progressive neurodegenerative pathology affecting hippocampus, temporal and/or frontal cerebral cortex lobes. An ideal reference gene for data normalization would be consistently expressed in all samples in any experimental condition, regardless of the tissue type and possible disease state whereas its quantitative expression should be comparable to that of the target gene [4,6]. In this context it is reasonable to conclude that there is not a universal reference gene suitable for different experimental conditions and that individual cases should contemplate a careful evaluation and validation of several reference genes prior to choosing the appropriate one [6,7]
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