Abstract
Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.
Highlights
Temporal lobe epilepsy (TLE) is one of the most common medically intractable neurological disorders
The pathogenesis of TLE involves abnormal neuronal reorganization occurring over a long period of time following a strong cerebral insult, such as status epilepticus (SE) [1,2]
Studies in patients or animal models of TLE show that the global gene expression pattern is significantly altered at time points more closely related to responses to either SE or cumulative chronic spontaneous recurrent seizures (SRS) [4,5,6,7,8,9]
Summary
Temporal lobe epilepsy (TLE) is one of the most common medically intractable neurological disorders. The pathogenesis of TLE involves abnormal neuronal reorganization occurring over a long period of time following a strong cerebral insult, such as status epilepticus (SE) [1,2]. These changes can include neurodegeneration, neurogenesis, gliosis, axonal damage or sprouting, dendritic plasticity, inflammation, reorganization of the extracellular matrix and alterations in the molecular structure of cellular components [3]. Epilepsy research has turned to the question of which regulator factors are involved in the reorganization of gene expression that accompanies the epileptic condition
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