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使用死后海马组织会干扰癫痫发生过程的差异基因表达分析。

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process.

作者信息

Born João Paulo Lopes, Matos Heloisa de Carvalho, de Araujo Mykaella Andrade, Castro Olagide Wagner, Duzzioni Marcelo, Peixoto-Santos José Eduardo, Leite João Pereira, Garcia-Cairasco Norberto, Paçó-Larson Maria Luisa, Gitaí Daniel Leite Góes

机构信息

Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

出版信息

PLoS One. 2017 Aug 4;12(8):e0182765. doi: 10.1371/journal.pone.0182765. eCollection 2017.

Abstract

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.

摘要

神经病理学研究经常使用尸检脑组织作为对照,以评估几种疾病中蛋白质或RNA水平的变化。在颞叶内侧癫痫(MTLE)中,几种基因在疾病的致痫阶段和慢性阶段上调或下调。鉴于几种基因转录本的死后变化可能会影响病例对照研究中变化的检测,我们评估了使用不同死后间隔(PMI)的尸检标本对毛果芸香碱(PILO)诱导的MTLE癫痫持续状态(SE)差异基因表达的影响。为此,我们选择了六个基因(Gfap、Ppia、Gad65、Gad67、Npy和Tnf-α),其在注射PILO的大鼠海马中的表达模式是众所周知的。最初,我们比较了死后立即采集海马的未处理大鼠(0小时-PMI)与死后6小时间隔(6小时-PMI)采集海马的大鼠的海马表达:6小时-PMI组中Npy和Ppia转录本增加,Tnf-α转录本减少(p<0.05)。然后,我们研究了这些与PMI相关的基因表达变化是否有可能掺假或掩盖在急性或慢性期安乐死的注射PILO的大鼠中获得的RT-qPCR结果。在急性组中,与0小时-PMI大鼠相比,Npy转录本显著更高,而Ppia转录本低于6小时-PMI组。当我们使用癫痫大鼠(慢性组)时,RT-qPCR结果仅在与6小时-PMI组比较时显示Tnf-α更高。总之,我们的研究表明PMI影响基因转录,并可掩盖PILO-SE模型癫痫发生过程中所见的基因转录变化。因此,为避免得出错误结论,我们强烈建议研究人员在其实验设计中考虑死后基因表达的变化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/83ea/5544225/959175e1ad07/pone.0182765.g001.jpg

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