Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?

Abstract

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex).Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder.Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively.Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

Evaluation of tumor necrosis factor (TNF)-α mRNA expression level and the rs1799964 polymorphism of the TNF-α gene in peripheral mononuclear cells of patients with inflammatory bowel diseases

“…The following primer pairs were used: Forward, 5-CTGAACTTCGGGGTGATCG-3 and reverse, 5-GCT TGG TGG TTT GCT ACG AC-3 for TNF-α; forward, 5-ACA ACC TTC TTG CAG CTC CTC-3 and reverse, 5-TGACCCATACCCACCATCAC-3 for β-actin (ACTB). The expression levels of all target genes were normalized against the expression of ACTB, which served as the endogenous control (20). The first stage, initial denaturation at 95˚C for 30 sec, was followed by a second 40-replication cycle (two-step cycling at 95˚C for 5 sec and 60˚C for 34 sec) and the third step (performed following creation of the PCR melting curve) was as follows: 95˚C for 15 sec, 60˚C for 60 sec and 95˚C for 15 sec.…”

Repetitive stress leads to impaired cognitive function that is associated with DNA hypomethylation, reduced BDNF and a dysregulated HPA axis

“…We chose β‐actin as reference gene as it has been repeatedly used in our laboratory and proved to be suitable for MR, GR and BDNF mRNA expression analysis (Mpofana et al, 2016). It has also been shown to have a stable mRNA expression in brain areas such as the striatum and hippocampus, and has been highly recommended by others (de Moura et al, 2014).…”

Effects of Herbal Compound (IMOD) on Behavior and Expression of Alzheimer's Disease Related Genes in Streptozotocin-Rat Model of Sporadic Alzheimer’s Disease

“…The normalization was done by Actb endogenous control. 23 , 24 Cycle threshold (Ct) values were used to calculate fold changes in gene expression between groups using REST 2009 software. P-values less than 0.01 for analysis by REST and in other analysis less than 0.05 were considered statistically significant.…”