通过微流控PCR对T细胞进行高度多重单细胞转录组分析

Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.

作者信息

Dominguez Maria, Roederer Mario, Chattopadhyay Pratip K

机构信息

Immunotechnology Section, Vaccine Research Center, NIH, 40 Convent Dr., Room 5612, Bethesda, MD, 20892, USA.

出版信息

Methods Mol Biol. 2017;1514:187-202. doi: 10.1007/978-1-4939-6548-9_15.

DOI:10.1007/978-1-4939-6548-9_15

PMID:27787802

Abstract

Recently, technologies have been developed to measure expression of 96 (or more) mRNA transcripts at once from a single cell. Here we describe methods and important considerations for use of Fluidigm's BioMark platform for multiplexed single cell gene expression. We describe how to qualify primer/probes, select genes to examine in 96-parameter panels, perform the reverse transcription/cDNA synthesis step, and operate the instrument. In addition, we describe data analysis considerations. This technology has enormous value for characterizing the heterogeneity of T-cells, thereby providing a useful tool for immune monitoring.

摘要

最近，已经开发出了能够同时从单个细胞中测量96个（或更多）mRNA转录本表达的技术。在这里，我们描述了使用Fluidigm公司的BioMark平台进行多重单细胞基因表达分析的方法和重要注意事项。我们阐述了如何鉴定引物/探针、选择要在96参数面板中检测的基因、进行逆转录/cDNA合成步骤以及操作仪器。此外，我们还描述了数据分析方面的注意事项。这项技术对于表征T细胞的异质性具有巨大价值，从而为免疫监测提供了一个有用的工具。