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Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

Nature Protocols volume 2, pages 739–752 (2007)Cite this article

Abstract

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (∼100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5–6 days.

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Figure 1: Schematic representation of the step-by-step protocol.
Figure 2: A mouth pipette.
Figure 3: A typical result of 2% agarose gel electrophoresis of the cDNAs amplified from 10 pg ES cell total RNA.
Figure 4: Amplification curves of genes in cDNAs from 10 pg ES cell total RNA during PCR amplification.
Figure 5: Anticipated representation of gene expressions after cDNA amplifications.
Figure 6: Preservation of transcript abundance through the gel purification step.
Figure 7: Typical results of IVT.
Figure 8: Preservation of transcript abundance through the IVT reaction.

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Acknowledgements

We thank all the members of our laboratory for their discussion of this study. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a PRESTO project grant from the Japan Science and Technology Agency.

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Authors and Affiliations

  1. Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    Kazuki Kurimoto, Yukihiro Yabuta, Yasuhide Ohinata & Mitinori Saitou

  2. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Hon-cho, Kawaguchi, Saitama, 332-0012, Japan

    Mitinori Saitou

  3. Laboratory of Molecular Cell Biology and Development, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan

    Mitinori Saitou

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  1. Kazuki Kurimoto
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Correspondence to Mitinori Saitou.

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Kurimoto, K., Yabuta, Y., Ohinata, Y. et al. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat Protoc 2, 739–752 (2007). https://doi.org/10.1038/nprot.2007.79

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