We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer’s disease (AD), Parkinson’s disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

我们检查了合并的正常脑脊液（CSF）样本和来自五个主要神经系统疾病的CSF的细胞外囊泡（EV）和RNA组成：阿尔茨海默氏病（AD），帕金森氏病（PD），低度神经胶质瘤（LGG），胶质母细胞瘤（GBM）和蛛网膜下腔出血（SAH），分别代表神经退行性疾病，癌症和严重的急性脑损伤。我们评估了：（I）纳米颗粒跟踪分析（NTA）和囊泡流式细胞仪（VFC）的电动汽车的大小和数量，（II）使用四个RNA分离试剂盒的RNA产量和纯度，（III）实验室内部和实验室之间RNA产量的复制（IV）通过逆转录-定量聚合酶链反应（RT-qPCR）和RNA测序（RNASeq）组成总RNA和EV RNA。NTA和VFC得出的CSF约为106 EVs /μL。与正常，AD和PD相比，脑瘤和SAH CSF含有更多的EV和RNA。RT-qPCR和RNASeq在总分数和EV分数中均确定了与正常CSF相关的疾病相关的microRNA和信使RNA（mRNA）种群。这项工作提出了选择相关的措施，以指导后续的CSF复制研究的设计。神经系统疾病的范围突出显示了由于疾病或采集地点而导致的总和EV RNA含量的变化，揭示了指导选择适当的方法和对照进行CSF研究的重要考虑因素。这项工作提出了选择相关的措施，以指导后续的CSF复制研究的设计。神经系统疾病的范围突出显示了由于疾病或采集部位而导致的总和EV RNA含量的变化，揭示了指导选择适当的方法和对照进行CSF研究的重要考虑因素。这项工作提出了选择相关的措施，以指导后续的重复性CSF研究的设计。神经系统疾病的范围突出显示了由于疾病或采集地点而导致的总和EV RNA含量的变化，揭示了指导选择适当的方法和对照进行CSF研究的重要考虑因素。