Comparison of human lower and upper entorhinal cortex layers gene expression. Homo sapiens

Specific vulnerability of neurons in the human entorhinal cortex has been associated with the onset of disease. Gene expression is analyzed to define the molecular characteristic of those neurons. Overall design: Human tissue collection and dissection. Brain samples were collected from four individuals with no clinical evidence of neurological disease and no neuropathological evidence of neurodegeneration. Tissue samples were obtained from the neurological tissue bank (UIPA) and from the Neurological Research Tissue Bank (BTIN, Madrid). The mean postmortem interval (PMI) of the tissue was 6 h and each subject died in hospital due to either cardiac or infectious diseases. The tissue was obtained according to local ethical and legal regulations concerning the use of human post-mortem tissue for biomedical research. Frozen tissue samples were collected from the entorhinal cortex [EC; Brodmann area (BA) 28], at coronal level 27 of the Atlas of Paxinos. Tissue samples corresponded to either upper (CES), lower (CEI) or the entire EC (CET). Two adjacent vertical columns comprising the full thickness of the EC were dissected under magnification with a Leica M50 stereomicroscope. One of them was then divided into two blocks, corresponding to CES and CEI, while the remaining column was processed as CET. RNA sample preparation. Cerebral tissue was homogenised in liquid nitrogen with a pestle and mortar, and the total RNA was isolated using the RNeasy Mini Kit and QIAshredder (Qiagen). The total RNA concentration and purity were determined using an Agilent2100 Bioanalyzer (Agilent Biotechnologies, Palo, Alto, CA) and by agarose gel electrophoresis. Subsequently, cDNA was synthesized using the One-Cycle cDNA Synthesis kit (Affymetrix), according to the protocol described in the Expression Analysis Technical Manual. Biotinylated cRNA probes were generated from each cDNA sample following the IVT Labeling kit instructions (Affymetrix), and the cRNA synthesized was purified with the GeneChip Sample Cleanup Module (Affymetrix). The concentration and purity of the biotinylated cRNA was determined using an Agilent2100 Bioanalyzer (Agilent Biotechnologies, Palo, Alto, CA) and by agarose gel electrophoresis.

人类内嗅皮层（entorhinal cortex, EC）神经元的特异性易感性与疾病发作密切相关。本研究通过分析基因表达，以明确该类神经元的分子特征。

总体实验设计：人体组织采集与解剖。研究共纳入4名无临床神经疾病证据、亦无神经退行性病变神经病理证据的个体，采集其脑样本。组织样本分别来源于神经组织库（UIPA）以及马德里神经研究组织库（BTIN）。该批组织的平均死后间隔时间（postmortem interval, PMI）为6小时，所有受试者均因心脏疾病或感染性疾病在医院离世。本组织获取流程严格遵循生物医学研究使用人体死后组织的当地伦理与法律法规要求。

从帕西诺斯（Paxinos）脑图谱冠状层面27处采集内嗅皮层[布罗德曼分区（Brodmann area, BA）28]的冰冻组织样本。组织样本分为三类：上部（CES）、下部（CEI）以及完整内嗅皮层（CET）。在Leica M50体式显微镜放大下，解剖得到两列相邻的、覆盖内嗅皮层全厚度的垂直组织柱：其中一列被切割为两块，分别对应CES与CEI样本，另一列则作为CET样本进行后续处理。

RNA样本制备流程：
将脑组织在液氮中用研钵和研杵研磨匀浆，随后使用RNeasy Mini试剂盒与QIAshredder（Qiagen公司）分离总RNA。通过Agilent2100生物分析仪（Agilent Biotechnologies, 帕洛阿尔托, 加利福尼亚州）与琼脂糖凝胶电泳检测总RNA的浓度与纯度。

随后，按照《表达分析技术手册》中的操作流程，使用One-Cycle cDNA Synthesis试剂盒（Affymetrix公司）合成cDNA。依照IVT标记试剂盒（Affymetrix公司）的说明书，从每份cDNA样本中制备生物素标记的cRNA探针，并通过GeneChip样本纯化模块（Affymetrix公司）纯化合成得到的cRNA。再次使用Agilent2100生物分析仪与琼脂糖凝胶电泳检测生物素标记cRNA的浓度与纯度。