Defining the nociceptor transcriptome

Unbiased 'omics' techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease. RNA-Seq was performed for 4 biological replicates from three different groups: intact DRG, acutely dissociated DRG and magnetically-purified DRG neurons. Differential expression was analyzed between acutely dissociated and MACS-dissociated samples to define the 'nociceptor transcriptome'.无偏倚组学（omics）技术（如下一代RNA测序（next generation RNA-sequencing)）可为生物系统的研究提供全然新颖的认知视角。然而，细胞异质性为这类数据集的分析与解读带来了显著阻碍。背根神经节（dorsal root ganglia, DRG）神经元是研究神经元损伤、再生与疼痛的重要模型。绝大多数研究者在探究细胞与分子功能时，会采用全神经节解离制备的样本。我们证实，当前制备这类样本的标准方法所得到的细胞混合物中，神经元占比仅为10%，其余细胞为卫星胶质细胞与其他非神经元细胞类型。通过一种新型磁纯化技术，我们可从成年组织中稳定获取纯度超95%的存活神经元，且这些神经元显著富集于表达电压门控离子通道（voltage gated ion channel）Nav1.8的小直径伤害性感受器群体中。我们借助全基因组RNA测序（genome-wide RNA-sequencing）对比了当前常用的（神经元占比10%）样本与纯（95%为伤害性感受器）样本，发现共有920个基因存在表达富集。这为背根神经节中小直径伤害性感受器神经元的分子组成提供了前所未有的解析，或可改写既往基于组织层面研究的解读结论，并为这一被广泛研究的细胞群体鉴定出多个全新标志物。我们预计，该技术具备操作简便、成本低廉、耗时较短的优势，有望得到广泛应用，从而大幅提升研究伤害性感受器在健康与疾病中作用的能力。本研究对三组样本的4份生物学重复开展了RNA测序：完整背根神经节样本、急性解离背根神经节样本，以及磁纯化背根神经节神经元样本。我们对急性解离样本与MACS解离样本进行了差异表达分析，以明确"伤害性感受器转录组"。