Soh Kah Teong, Wallace Paul K
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263, USA.
Methods Mol Biol. 2018;1678:49-77. doi: 10.1007/978-1-4939-7346-0_4.
The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.
mRNA和蛋白质的系统性调控支配着我们细胞复杂且相互交织的生物学功能。传统上，诸如DNA微阵列和RNA测序等转录组技术已被用于识别、表征和分析基因表达数据。然而，这些都被视为批量方法，因为它们无法在单细胞水平上测量基因表达，除非细胞预先进行分选。分支DNA是一种基于流式细胞术的检测平台，最近已被开发用于在单细胞水平上测量mRNA。该系统最初源自显微镜技术，目前已进行了改进，以实现与使用与荧光染料偶联的单克隆抗体检测表面和细胞内抗原的兼容性，从而允许同时检测mRNA和蛋白质。与用于定量mRNA的传统或标准方法相比，分支DNA方法具有多种优势，例如：（a）在单个细胞基础上检测特定mRNA；（b）当蛋白质测量在技术上不可行（即不存在高质量抗体）或表位不可评估时的替代检测工具；（c）将mRNA分析与蛋白质分析相关联。与早期通过流式细胞术测量核酸的尝试相比，分支DNA测定中应用的杂交温度要低得多，从而保留了细胞结构的完整性以便进一步表征。它还大大提高了特异性和灵敏度。在这里，我们提供了在模型系统中使用分支DNA方法通过流式细胞术关联CD8 mRNA和CD8蛋白表达的详细操作说明。
