title: A microfluidic processor for gene expression profiling of single human embryonic stem cells.
abstract: 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.
authors: Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. jzhong@usc.edu
authors: Jiang F
authors: JF
authors: Zhong
authors: Yan
authors: Y
authors: Chen
authors: Joshua S
authors: JS
authors: Marcus
authors: Axel
authors: A
authors: Scherer
authors: Stephen R
authors: SR
authors: Quake
authors: Clive R
authors: CR
authors: Taylor
authors: Leslie P
authors: LP
authors: Weiner
mesh_terms: D000818:Animals; D002452:Cell Count; D018076:DNA, Complementary / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D051379:Mice; D046210:Microfluidic Analytical Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase