Selection of Candidate Housekeeping Genes for Normalization in Human Postmortem Brain Samples

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.

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

“…This result is consistent with that of a previous study [35]; however, the efficacy of the other conventional references, ACTB and B2M, could not be established. In fact, there is a lack of consensus about the determination of optimal reference genes in postmortem human tissues [35,36,46,47], although most experiments were performed under MIQE Guidelines [48,49]. The different results may owe to varied requirements and conditions in individual studies.…”

“…a Expression stability of genes from the least stable (left) to most stable (right). b Pairwise variation analysis to determine the optimal number of reference genes for normalization [36], could also be calculated by GenEx Standard software and showed that the Acc.S.D. (0.092) was the lowest when eight reference genes were used (Fig.…”

The miR-15/107 Family of microRNA Genes Regulates CDK5R1/p35 with Implications for Alzheimer’s Disease Pathogenesis

“…n = 5, mean ± s.d. *p < 0.05 **p < 0.01, Student's t test genes, as suggested by Penna and colleagues [33], as well as against the levels of the neuronal marker SYP, in order to normalize gene expression taking into account the neuronal population only [34].…”

Real-Time PCR for Gene Expression Analysis

“…The cyclophilin A gene [peptidylprolyl isomerase A gene (PPIA)] is recommended as a housekeeping gene for gene expression studies in atopic human airway epithelial cells (AEC) of asthmatics (He et al, 2008). Penna et al suggested that the use of two reference genes [Eukaryotic translation initiation factor 4A2 (EIF4A2) and Cytochrome c-1 (CYC1)] is proper for the normalization of the RT-qPCR data in human brain tissues (Penna et al, 2011). Pfister et al demonstrated that the ribosomal protein L37A (RPL37A) is the most ideal housekeeping gene in meningiomas and their normal control tissue arachnoidea, dura mater and normal brain.…”