Thermo Fisher Scientific, 3380 Central Expressway, Santa Clara, CA 95051, United States.
Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, United States.
Methods. 2018 Feb 1;134-135:136-148. doi: 10.1016/j.ymeth.2017.12.016. Epub 2017 Dec 19.
Analysis of RNA expression in mixed cell populations often requires laborious and costly cell sorting. Here we describe a flow cytometric assay that combines antibody staining and in situ hybridization for multi-parametric analysis of single cells. This method, referred to as the PrimeFlow™ RNA Assay, enables simultaneous detection of protein markers and RNA targets in mixed cell populations. Both coding and non-coding RNA sequences can be measured with a limit of detection of approximately 10 copies of mRNA and 20 copies of microRNA per cell. In this study, we used mouse bone marrow-derived macrophages to demonstrate that our method allows for analysis of the activation and polarization status of cells using expression patterns of protein and RNA. We then performed analysis of four cell subsets of mouse resident peritoneal cells and showed that the two macrophage populations present in this compartment are relatively heterogeneous in terms of expression of two M2 markers: Arg1, Retnla, and a B-cell attractant chemokine Cxcl13. In addition, we profiled the expression of a panel of microRNA in the four peritoneal cell subsets, showing that the assay can be readily adapted to parallel, high-throughput screening of multiple cell populations. This new method allows for single cell analysis of multiple RNA targets without the need for cell sorting, enables direct correlation between RNA and protein expression, and promises to accelerate biomarker and drug discovery.
分析混合细胞群中的 RNA 表达通常需要繁琐且昂贵的细胞分选。在这里，我们描述了一种流式细胞术检测方法，该方法结合抗体染色和原位杂交，可对单细胞进行多参数分析。这种方法被称为 PrimeFlow™ RNA 检测法，可在混合细胞群中同时检测蛋白质标志物和 RNA 靶标。编码 RNA 和非编码 RNA 序列都可以被测量，其检测限大约为每个细胞 10 个 mRNA 拷贝和 20 个 microRNA 拷贝。在本研究中，我们使用小鼠骨髓来源的巨噬细胞证明，我们的方法可以使用蛋白质和 RNA 的表达模式分析细胞的激活和极化状态。然后，我们对小鼠固有腹膜细胞的四个细胞亚群进行了分析，结果表明，该隔室中存在的两种巨噬细胞群体在两个 M2 标志物的表达上存在相对异质性：Arg1、Retnla 和 B 细胞趋化因子 Cxcl13。此外，我们对四个腹膜细胞亚群中的一组 microRNA 的表达进行了分析，表明该检测法可轻松适应多个细胞群的并行、高通量筛选。这种新方法可在无需细胞分选的情况下对多个 RNA 靶标进行单细胞分析，实现 RNA 和蛋白质表达之间的直接关联，并有望加速生物标志物和药物发现。
