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
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Kobayashi, H. Mizuno, I. Imayoshi, C. Furusawa, K. Shirahige, and R. Kageyama The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells Genes & Dev., August 15, 2009; 23(16): 1870 - 1875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rossant and P. P. L. Tam Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse Development, March 1, 2009; 136(5): 701 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. May, R. Kirchner, H. Muller, P. Hartmann, N. El Hajj, A. Tresch, U. Zechner, W. Mann, and T. Haaf Multiplex RT-PCR Expression Analysis of Developmentally Important Genes in Individual Mouse Preimplantation Embryos and Blastomeres Biol Reprod, January 1, 2009; 80(1): 194 - 202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Plusa, A. Piliszek, S. Frankenberg, J. Artus, and A.-K. Hadjantonakis Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst Development, September 15, 2008; 135(18): 3081 - 3091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kawaguchi, T. Ikawa, T. Kasukawa, H. R. Ueda, K. Kurimoto, M. Saitou, and F. Matsuzaki Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis Development, September 15, 2008; 135(18): 3113 - 3124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tougan, D. Okuzaki, and H. Nojima Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification Nucleic Acids Res., September 1, 2008; 36(15): e92 - e92. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kurimoto, Y. Yabuta, Y. Ohinata, M. Shigeta, K. Yamanaka, and M. Saitou Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice Genes & Dev., June 15, 2008; 22(12): 1617 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-E. Dietrich and T. Hiiragi Stochastic patterning in the mouse pre-implantation embryo Development, December 1, 2007; 134(23): 4219 - 4231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. Gallardo, G. B. John, L. Shirley, C. M. Contreras, E. A. Akbay, J. M. Haynie, S. E. Ward, M. J. Shidler, and D. H. Castrillon Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse Genetics, September 1, 2007; 177(1): 179 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Tang, M. Kaneda, D. O'Carroll, P. Hajkova, S. C. Barton, Y. A. Sun, C. Lee, A. Tarakhovsky, K. Lao, and M. A. Surani Maternal microRNAs are essential for mouse zygotic development Genes & Dev., March 15, 2007; 21(6): 644 - 648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kaji, J. Nichols, and B. Hendrich Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells Development, March 15, 2007; 134(6): 1123 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Hartmann and C. A. Klein Gene expression profiling of single cells on large-scale oligonucleotide arrays Nucleic Acids Res., December 4, 2006; 34(21): e143 - e143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yabuta, K. Kurimoto, Y. Ohinata, Y. Seki, and M. Saitou Gene Expression Dynamics During Germline Specification in Mice Identified by Quantitative Single-Cell Gene Expression Profiling Biol Reprod, November 1, 2006; 75(5): 705 - 716. [Abstract] [Full Text] [PDF]
|
|