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Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity.

Journal article
Authors Emma Jonasson
Soheila Dolatabadi
Pierre Åman
Anders Ståhlberg
Published in Cells
Volume 9
Issue 3
ISSN 2073-4409
Publication year 2020
Published at Sahlgrenska Cancer Center
Department of Laboratory Medicine
Wallenberg Centre for Molecular and Translational Medicine
Language en
Links dx.doi.org/10.3390/cells9030759
www.ncbi.nlm.nih.gov/entrez/query.f...
Keywords cell heterogeneity, sarcoma, single-cell analysis, total mRNA level, transcriptome size
Subject categories Cancer and Oncology, Cell and Molecular Biology

Abstract

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.

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