Seleção de genes de referência para estudos de expressão gênica utilizando qPCR em um modelo animal de neurodegeneração induzida por estreptozotocina

• Ano de defesa: 2016
• Autor(a) principal: Wohlgemuth, Kenny Nieradka
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Positivo; Brasil; Pós-Graduação; Programa de Pós-Graduação em Biotecnologia Industrial; UP
• Palavras-chave em Português: Biotecnologia; Regulação de expressão gênica; Genes reguladores; Hipocampo; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: https://repositorio.cruzeirodosul.edu.br/handle/123456789/2608

The reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) is currently one of the techniques most commonly used in studies evaluating gene expression. Due to some peculiarities inherent to the technique, normalization methods are required to achieve reliable data and accurate results. The method most commonly used for normalization of RT-qPCR assays is using reference genes, which should be unaffected by the experimental conditions.Therefore, validation of suitable reference genes is an essential step in studies aimed at analyzing gene expression of specific targets. The objective of this study was to identify the most stable reference to be used for optimal normalization in an experimental rat model of neurodegeneration. We used the hippocampus tissue of Wistar rats that were previously injected intracerebroventricularly with a drug called streptozotocin, which is known to induce molecular changes similar to those occurring in Alzheimer's disease. This animal model is very important to improve the knowledge of molecular and biochemical changes that occur during this disease. The reference genes chosen for analysis were the most commonly used for normalization of RT-qPCR studies in the central nervous system: 18s subunit ribosomal RNA (18s RNA), beta-actin (Actb), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine fosfoborisil transferase 1 (Hprt1) and ribosomal protein L13a (Rpl13a). Three softwares were used, geNorm, NormFinder BestKeeper, to evaluate the stability of candidate reference genes. The programs indicated Rpl13a as the more stable reference gene in the hippocampus of rats injected with streptozotocin. Therefore, we suggest the use of Rpl13a as reference gene in studies using RT-qPCR will allow accurate and reliable normalization of gene expression data in this animal model of neurodegeneration.