Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.
Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.
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DOI：
10.1371/journal.pone.0100529
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Gitaí DL
中科院分区：
文献类型：
--
作者：
de Araújo MA;Marques TE;Taniele-Silva J;Souza FM;de Andrade TG;Garcia-Cairasco N;Paçó-Larson ML;Gitaí DL
实时定量 RT-PCR (qPCR) 因其敏感性和特异性而成为分析 miRNA 表达的最强大技术之一。然而，在此类分析中，需要合适的标准化器以确保基因表达不受实验条件的影响。据我们所知，尚无研究报道对癫痫发生过程中 miRNA qPCR 的合适参考基因进行了详细的鉴定和验证。在这里，我们使用内侧颞叶癫痫 (MTLE) 的毛果芸香碱 (PILO) 模型，研究了五个潜在的参考基因，并使用 geNorm 和 NormFinder 软件进行稳定性表达分析。作为一种验证策略，我们使用每个候选参考基因来测量 PILO 诱导的 microRNA-146a 水平的变化，该基因在 PILO 注射模型中的表达模式变化是已知的。我们的结果表明 U6SnRNA 和 SnoRNA 是最稳定的候选参考基因。通过geNorm分析，标准化因子优选地应包含至少两个最佳候选参考基因（snoRNA和U6SnRNA）。事实上，当使用参考基因的最佳组合进行标准化时，发现慢性阶段的 microRNA-146a 转录物显着增加，这与不同模型中报告的模式一致。相反，当单独使用参考基因进行标准化时，我们未能检测到癫痫大鼠海马中 microRNA-146a 基因的上调。这里提供的数据支持 snoRNA 和 U6SnRNA 的组合是我们测试的癫痫发生不同阶段基因表达精确标准化所需的最低限度。
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.
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影响因子：
7.3
作者：
Chugh, Pauline;Dittmer, Dirk P.
通讯作者：
Dittmer, Dirk P.
影响因子：
3.5
作者：
Bustin, SA
通讯作者：
Bustin, SA
正常人组织中microRNA表达谱的表征。
影响因子：
4.4
作者：
Liang, Yu;Ridzon, Dana;Wong, Linda;Chen, Caifu
通讯作者：
Chen, Caifu
影响因子：
3.4
作者：
Becker, AJ;Chen, J;Blümcke, I
通讯作者：
Blümcke, I
影响因子：
1.7
作者：
Hill JM;Zhao Y;Clement C;Neumann DM;Lukiw WJ
通讯作者：
Lukiw WJ