Published online 17 March 2006
X1 microarray |
An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis
1Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 2Functional Genomics Subunit, Center for Developmental Biology, RIKEN Kobe Institute 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 3Laboratory for Systems Biology, Center for Developmental Biology, RIKEN Kobe Institute 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 4Department of BioScience, Tokyo University of Agriculture Setagaya-ku, Tokyo 156-8502, Japan 5Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan 6Laboratory of Molecular Cell Biology and Development, Graduate School of Biostudies, Kyoto University Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
*To whom correspondence should be addressed. Tel: +81 78 306 3376; Fax: +81 78 306 3377; Email: saitou{at}cdb.riken.jp
Received January 17, 2006. Revised February 3, 2006. Accepted February 23, 2006.
A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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