Porter Joshua R, Telford William G, Batchelor Eric
Laboratory of Pathology, Center for Cancer Research, National Cancer Institute.
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute.
J Vis Exp. 2017 Feb 25(120):55219. doi: 10.3791/55219.
Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.
对大量细胞群体进行基因表达测量可能会掩盖这些群体中单个细胞相当大的转录组变异。单细胞基因表达测量有助于评估基因表达中噪声的作用，识别基因对表达中的相关性，并揭示对刺激有不同反应的细胞亚群。在这里，我们描述了一种用于测量从组织培养中生长的群体中分离出的单个哺乳动物细胞中多达96个基因表达的方法。通过荧光激活细胞分选（FACS）将细胞分选到裂解缓冲液中，然后将感兴趣的mRNA物种进行逆转录和扩增。然后使用微流控实时PCR仪测量基因表达，该仪器一次可对多达96个样本进行多达96次qPCR检测。我们还描述了PCR扩增子标准品的生成和使用，以估计每个转录本的绝对数量。与其他测量单细胞基因表达的方法相比，这种方法比RNA荧光原位杂交（FISH）能够定量更多不同的转录本，且成本低于RNA测序（RNA-Seq）。
