Sahlgrenska Cancer Center, Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, SE-405 30, Gothenburg, Sweden.
Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, SE-405 30, Gothenburg, Sweden.
Cells. 2020 Mar 19;9(3):759. doi: 10.3390/cells9030759.
Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions.
单细胞分析能够详细地描述细胞类型、基因型、细胞状态、时间变化和微环境等方面的分子特征。这些研究通常包括对单个基因和基因网络的分析。由于细胞类型、细胞大小和细胞周期状态等重要因素，细胞之间的总 RNA 量也存在差异。然而，目前缺乏简单而灵敏的方法来定量总 RNA，特别是 mRNA。在这里，我们开发了一种基于全球反转录和定量 PCR 的方法来定量单细胞中的总 mRNA 水平。稀释 RNA 和分选细胞的标准曲线分析显示，该方法具有较宽的动态范围、高重复性和优异的灵敏度。对三种肉瘤细胞系和人成纤维细胞的单细胞分析表明，存在细胞类型的差异，总 mRNA 水平呈对数正态分布，并且细胞之间的总 mRNA 水平差异高达 8 倍。该方法可以很容易地与靶向或全局基因表达谱相结合，为在单个基因水平和全局水平上研究细胞异质性提供了新的手段。该方法可用于研究健康和病理条件下总 mRNA 量变化的生物学意义。
