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