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.

  • Article
  • Published:

Profiling alternative splicing on fiber-optic arrays

Nature Biotechnology volume 20, pages 353–358 (2002)Cite this article

Abstract

The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10–100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The technology platform and experimental strategy.
Figure 2: Detection of splicing changes in transfected cells.
Figure 3: Specificity and sensitivity of the RASL-PCR assay.
Figure 4: Characterization of multiplexed RASL-PCR.
Figure 5: Cell type–specific gene expression and alternative splicing analyzed by RASL-PCR and RT-PCR.
Figure 6: Potential autocrine signaling in human cancer cell lines.

Similar content being viewed by others

References

  1. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    CAS  PubMed  Google Scholar 

  2. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    CAS  PubMed  Google Scholar 

  3. Graveley, B.R. Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 17, 100–107 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Schmucker, D. et al. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671–684 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Lopez, A.J. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu. Rev. Genet. 32, 279–305 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Smith, C.W. & Valcárcel, J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Black, D.L. Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367–370 (2000).

    CAS  PubMed  Google Scholar 

  8. Shoemaker, D.D. et al. Experimental annotation of the human genome using microarray technology. Nature 409, 922–927 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Hu, G.K. et al. Predicting splice variant from DNA chip expression data. Genome Res. 11, 1237–1245 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Michael, K.L., Taylor, L.C., Schultz, S.L. & Walt, D.R. Randomly ordered addressable high-density optical sensor arrays. Anal. Chem. 70, 1242–1248 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Ferguson, J.A., Steemers, F.J. & Walt, D.R. High-density fiber-optic DNA random microsphere array. Anal. Chem. 72, 5618–5624 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Walt, D.R. Bead-based fiber-optic arrays. Science 287, 451–452 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Gerry, N.P. et al. Universal DNA microarray method for multiplex detection of low abundance point mutations. J. Mol. Biol. 292, 251–262 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Fan, J.B. et al. Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res. 10, 853–860 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science 241, 1077–1080 (1988).

    Article  CAS  PubMed  Google Scholar 

  16. Nilsson, M., Antson, D.O., Barbany, G. & Landegren, U. RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 29, 578–581 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kleppe, K., Van de Sande, J.H. & Khorana, H.G. Polynucleotide ligase-catalyzed joining of deoxyribo-oligonucleotides on ribopolynucleotide templates and of ribo-oligonucleotides on deoxyribopolynucleotide templates. Proc. Natl. Acad. Sci. USA 67, 68–73 (1970).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Harper, J.E. & Manley, J.L. Multiple activities of the human splicing factor ASF. Gene Expression 2, 19–29 (1992).

    CAS  PubMed  Google Scholar 

  19. Cáceres, J.F., Stamm, S., Helfman, D.M. & Krainer, A.R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science 265, 1706–1709 (1994).

    Article  PubMed  Google Scholar 

  20. James, M.C. & Peters, G. Alternative product of the p16/CKDN2A locus connects the Rb and p53 tumor suppressors. Prog. Cell Cycle Res. 4, 71–81 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Liggett, W.H. Jr., & Sidransky, D. Role of the p16 tumor suppressor gene in cancer. J. Clin. Oncol. 16, 1197–1206 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Jiang, Z.H. & Wu, J.Y. Alternative splicing and programmed cell death. Proc. Soc. Exp. Biol. Med. 220, 64–72 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Trowbridge, I.S. & Thomas, M.L. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12, 85–116 (1994).

    Article  CAS  PubMed  Google Scholar 

  24. Miki, T. et al. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251, 72–75 (1991).

    Article  CAS  PubMed  Google Scholar 

  25. Xu, X., Weinstein, M., Li, C. & Deng, C. Fibroblast growth factor receptors (FGFRs) and their roles in limb development. Cell Tiss. Res. 296, 33–43 (1999).

    Article  CAS  Google Scholar 

  26. De Moerlooze, L. et al. An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal–epithelial signaling during mouse organogenesis. Development 127, 483–492 (2000).

    CAS  PubMed  Google Scholar 

  27. Hajihosseini, M.K., Wilson, S., De Moerlooze, L. & Dickson, C. A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes. Proc. Natl. Acad. Sci. USA 98, 3855–3860 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Miki, T. et al. Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA 89, 246–250 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yan, G., Fukabori, Y., McBride, G., Nikolaropolous, S. & McKeehan, W.L. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)–FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol. Cell. Biol. 13, 4513–4522 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Carstens, R.P., Eaton, J.V., Krigman, H.R., Walther, P.J. & Garcia-Blanco, M.A. Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in human prostate cancer. Oncogene 15, 3059–3065 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Watanabe, M., Ishiwata, T., Nishigai, K., Moriyama, Y. & Asano, G. Overexpression of keratinocyte growth factor in cancer cells and enterochromaffin cells in human colorectal cancer. Pathol. Intl. 50, 363–372 (2000).

    Article  CAS  Google Scholar 

  32. Taniguchi, F. et al. Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation. Mol. Reprod. Dev. 50, 54–62 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Taniguchi, F. et al. Keratinocyte growth factor in the promotion of human chorionic gonadotropin production in human choriocarcinoma cells. Am. J. Obstet. Gynecol. 182, 692–698 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Y.-S. Kwon and other members of the Fu laboratory for their assistance, E. Chudin from Illumina for statistical analyses, and S. Goodison, C. Nelson, H. Li, C. Buckmaster, A. Boyer, M. Ricote, J. Brown, C. Snyder, and E. Brinkman-van der Linden for their gifts of cell lines. D. Che, F. Garcia, and J. Haas provided expert assistance with imaging systems and image analysis. X-D. F. is a Scholar of the Leukemia and Lymphoma Society. This work was supported by grants from the American Cancer Society (J.M.Y) and National Institutes of Health (X-D. F. and M.C.).

Author information

Author notes

  1. Jian-Bing Fan

    Present address: Illumina, Inc., 9885 Towne Centre Drive, San Diego, 92121, CA

  2. Joanne M. Yeakley

    Present address: Illumina, Inc., 9885 Towne Centre Drive, San Diego, CA, 92121

  3. Lin Luo

    Present address: Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA, 92121

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, CA

    Joanne M. Yeakley, Zhen Ye & Xiang-Dong Fu

  2. Illumina, Inc., 9885 Towne Centre Drive, San Diego, 92121, CA

    Dennis Doucet, Lin Luo, Eliza Wickham & Mark S. Chee

Authors
  1. Joanne M. Yeakley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian-Bing Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dennis Doucet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lin Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eliza Wickham
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhen Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mark S. Chee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiang-Dong Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jian-Bing Fan or Xiang-Dong Fu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yeakley, J., Fan, JB., Doucet, D. et al. Profiling alternative splicing on fiber-optic arrays. Nat Biotechnol 20, 353–358 (2002). https://doi.org/10.1038/nbt0402-353

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0402-353

This article is cited by

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