Zhong, Jiang F. and Chen, Yan and Marcus, Joshua S. and Scherer, Axel and Quake, Stephen R. and Taylor, Clive R. and Weiner, Leslie P. (2008) A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab on a Chip, 8 (1). pp. 68-74. ISSN 1473-0197 http://resolver.caltech.edu/CaltechAUTHORS:ZHOloac08
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The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These cell population averaging data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.
|Additional Information:||This journal is © The Royal Society of Chemistry 2008. Received 17th August 2007, Accepted 18th October 2007. First published on the web 2nd November 2007. This work was supported by grants from the National Institutes of Health (NIH 1RO1 HG002644-01A1) and the Nancy Davis Foundation for Multiple Sclerosis. The authors thank Dr Richard Vestewig for his suggestions and proof reading of the manuscript. The authors also thank the laboratory of Dr Melvin Simon and the laboratory of Dr Vijay Kalra for their assistance in real-time PCR.|
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|Deposited On:||30 Jan 2008|
|Last Modified:||26 Dec 2012 09:49|
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