Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.
Mykaella Andrade de Araújo, Thalita Ewellyn Batista Sales Marques, Jamile Taniele-Silva, Fernanda Maria de Araújo Souza, Tiago Gomes de Andrade, Norberto Garcia-Cairasco, Maria Luisa Paçó-Larson, Daniel Leite Góes Gitaí
Author Information
Mykaella Andrade de Araújo: Department of Cell, Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.
Thalita Ewellyn Batista Sales Marques: Department of Cell, Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.
Jamile Taniele-Silva: Department of Cell, Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.
Fernanda Maria de Araújo Souza: Department of Cell, Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.
Tiago Gomes de Andrade: Campus Arapiraca, Federal University of Alagoas, Maceió, Alagoas, Brazil.
Norberto Garcia-Cairasco: Department of Physiology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Maria Luisa Paçó-Larson: Department of Cellular and Molecular Biology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Daniel Leite Góes Gitaí: Department of Cell, Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.
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.
