Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

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.

Similar content being viewed by others

References

  1. Lockhart, D.J. et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14, 1675–1680 (1996).

    Article  CAS  Google Scholar 

  2. Adams, M.D. et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–1656 (1991).

    Article  CAS  Google Scholar 

  3. Boguski, M.S., Lowe, T.M. & Tolstoshev, C.M. dbEST—database for “expressed sequence tags”. Nat. Genet. 4, 332–333 (1993).

    Article  CAS  Google Scholar 

  4. Velculescu, V.E., Zhang, L., Vogelstein, B. & Kinzler, K.W. Serial analysis of gene expression. Science 270, 484–487 (1995).

    Article  CAS  Google Scholar 

  5. Brady, G., Barbara, M. & Iscove, N. Representative in vitro cDNA amplification from individual hemopoietic cells and colonies. Methods Mol. Cell. Biol. 2, 17–25 (1990).

    CAS  Google Scholar 

  6. Brady, G. & Iscove, N.N. Construction of cDNA libraries from single cells. Methods Enzymol. 225, 611–623 (1993).

    Article  CAS  Google Scholar 

  7. Van Gelder, R.N. et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663–1667 (1990).

    Article  CAS  Google Scholar 

  8. Eberwine, J. et al. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. USA 89, 3010–3014 (1992).

    Article  CAS  Google Scholar 

  9. Saito, H., Kubota, M., Roberts, R.W., Chi, Q. & Matsunami, H. RTP family members induce functional expression of mammalian odorant receptors. Cell 119, 679–691 (2004).

    Article  CAS  Google Scholar 

  10. Klur, S., Toy, K., Williams, M.P. & Certa, U. Evaluation of procedures for amplification of small-size samples for hybridization on microarrays. Genomics 83, 508–517 (2004).

    Article  CAS  Google Scholar 

  11. Ji, W., Zhou, W., Gregg, K., Lindpaintner, K. & Davis, S. A method for gene expression analysis by oligonucleotide arrays from minute biological materials. Anal. Biochem. 331, 329–339 (2004).

    Article  CAS  Google Scholar 

  12. Iscove, N.N. et al. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol. 20, 940–943 (2002).

    Article  CAS  Google Scholar 

  13. Kurimoto, K. et al. An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res. 34, e42 (2006).

    Article  Google Scholar 

  14. Tietjen, I. et al. Single-cell transcriptional analysis of neuronal progenitors. Neuron 38, 161–175 (2003).

    Article  CAS  Google Scholar 

  15. Chazaud, C., Yamanaka, Y., Pawson, T. & Rossant, J. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2–MAPK pathway. Dev. Cell 10, 615–624 (2006).

    Article  CAS  Google Scholar 

  16. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. & Hunter, C.P. Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889–900 (2003).

    Article  CAS  Google Scholar 

  17. Adams, M.D. et al. The genome sequence of Drosophila melanogaster . Science 287, 2185–2195 (2000).

    Article  Google Scholar 

  18. Church, R.B. & Robertson, F.W. Biochemical analysis of genetic differences in the growth of Drosophila . Genet. Res. 7, 383–407 (1966).

    Article  CAS  Google Scholar 

  19. Rahner, C., Fukuhara, M., Peng, S., Kojima, S. & Rizzolo, L.J. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J. Cell Sci. 117, 3307–3318 (2004).

    Article  CAS  Google Scholar 

  20. Jost, J.P., Ohno, T., Panyim, S. & Schuerch, A.R. Appearance of vitellogenin mRNA sequences and rate of vitellogenin synthesis in chicken liver following primary and secondary stimulation by 17 beta-estradiol. Eur. J. Biochem. 84, 355–361 (1978).

    Article  CAS  Google Scholar 

  21. Brown, D.D. & Gurdon, J.B. Absence of ribosomal RNA aynthesis in the anucleolate mutant of Xenopus laevis . Proc. Natl. Acad. Sci. USA 51, 139–146 (1964).

    Article  CAS  Google Scholar 

  22. Dulac, C. & Axel, R. A novel family of genes encoding putative pheromone receptors in mammals. Cell 83, 195–206 (1995).

    Article  CAS  Google Scholar 

  23. Saitou, M., Barton, S.C. & Surani, M.A. A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002).

    Article  CAS  Google Scholar 

  24. Chiang, M.K. & Melton, D.A. Single-cell transcript analysis of pancreas development. Dev. Cell 4, 383–393 (2003).

    Article  CAS  Google Scholar 

  25. Yabuta, Y., Kurimoto, K., Ohinata, Y., Seki, Y. & Saitou, M. Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling. Biol. Reprod. 75, 705–716 (2006).

    Article  CAS  Google Scholar 

  26. Kamme, F. et al. Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity. J. Neurosci. 23, 3607–3615 (2003).

    Article  CAS  Google Scholar 

  27. Seipp, S. & Buselmaier, W. Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis. Neurosci. Lett. 182, 91–94 (1994).

    Article  CAS  Google Scholar 

  28. Chambers, I. et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

    Article  CAS  Google Scholar 

  29. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).

    Article  CAS  Google Scholar 

  30. Avilion, A.A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003).

    Article  CAS  Google Scholar 

  31. Takahashi, K., Mitsui, K. & Yamanaka, S. Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423, 541–545 (2003).

    Article  CAS  Google Scholar 

  32. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  CAS  Google Scholar 

  33. Sato, M. et al. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech. Dev. 113, 91–94 (2002).

    Article  CAS  Google Scholar 

  34. Brennan, J. et al. Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411, 965–969 (2001).

    Article  CAS  Google Scholar 

  35. Rogers, M.B., Hosler, B.A. & Gudas, L.J. Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes. Development 113, 815–824 (1991).

    CAS  PubMed  Google Scholar 

  36. Laible, G. et al. Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J. 16, 3219–3232 (1997).

    Article  CAS  Google Scholar 

  37. Kawakami, A. et al. Identification and functional characterization of a TIA-1-related nucleolysin. Proc. Natl. Acad. Sci. USA 89, 8681–8685 (1992).

    Article  CAS  Google Scholar 

  38. Astigiano, S. et al. Changes in gene expression following exposure of nulli-SCCl murine embryonal carcinoma cells to inducers of differentiation: characterization of a down-regulated mRNA. Differentiation 46, 61–67 (1991).

    Article  CAS  Google Scholar 

  39. MacGregor, G.R., Zambrowicz, B.P. & Soriano, P. Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 121, 1487–1496 (1995).

    CAS  PubMed  Google Scholar 

  40. Safrany, G. & Perry, R.P. Characterization of the mouse gene that encodes the delta/YY1/NF-E1/UCRBP transcription factor. Proc. Natl. Acad. Sci. USA 90, 5559–5563 (1993).

    Article  CAS  Google Scholar 

  41. Morrisey, E.E., Ip, H.S., Lu, M.M. & Parmacek, M.S. GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev. Biol. 177, 309–322 (1996).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Kazuki Kurimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yukihiro Yabuta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yasuhide Ohinata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mitinori Saitou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mitinori Saitou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.79

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing