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Amine-modified random primers to label probes for DNA microarrays

Nature Biotechnology volume 20, pages 738–742 (2002)Cite this article

Abstract

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues1,2,3. The methods most commonly used to label probes for microarray studies require a minimum of 20 μg of total RNA or 2 μg of poly(A) RNA4,5. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described6,7,8,9. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 μg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5′ ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.

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Figure 1: Results obtained with three probe labeling methods.
Figure 2: Labeling of the same RNA sample with Cy5 and Cy3.

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References

  1. DeRisi, J. et al. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat. Genet. 14, 457–460 (1996).

    Article  CAS  Google Scholar 

  2. Heller, R.A. et al. Discovery and analysis of inflammatory disease–related genes using cDNA microarray. Proc. Natl. Acad. Sci. USA 94, 2150–2155 (1997).

    Article  CAS  Google Scholar 

  3. Wen, X. et al. Large-scale temporal gene expression mapping of central nervous system development. Proc. Natl. Acad. Sci. USA 95, 334–339 (1998).

    Article  CAS  Google Scholar 

  4. Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 (1995).

    Article  CAS  Google Scholar 

  5. Duggan, D., Bitter, M., Chen, Y., Meltzer, P. & Trent, J. Expression profiling using cDNA microarrays. Nat. Genet. Suppl. 21, 10–14 (1999)

    Article  CAS  Google Scholar 

  6. Wang, E., Miller, L.D., Ohnmacht, G.A., Liu, E.T. & Marincola, F.M. High-fidelity mRNA amplification for gene profiling. Nat. Biotechnol. 18, 457–459 (2000).

    Article  CAS  Google Scholar 

  7. Stears, R.L., Getts, R.C. & Gullans, S.R. A novel, sensitive detection system for high-density microarrays using dendrimer technology. Physiol. Genomics 3, 93–99 (2000).

    Article  CAS  Google Scholar 

  8. Baelde, H.J. et al. High quality RNA isolation from tumors with low cellularity and high extracellular matrix component for cDNA microarrays: application to chondrosarcoma. J. Clin. Pathol. 54, 778–782 (2001).

    Article  CAS  Google Scholar 

  9. Wong, K.-K., Cheng, R.S. & Mok, S.C. Identification of differentially expressed genes from ovarian cancer cells by MICROMAX cDNA microarray system. Biotechniques 30, 670–675 (2001).

    Article  CAS  Google Scholar 

  10. Eisen, M.B. & Brown, P.O. DNA arrays for analysis of gene expression. Methods Enzymol. 303, 179–205 (1999).

    Article  CAS  Google Scholar 

  11. Lashkari, D.A. et al. Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proc. Natl. Acad. Sci. USA 94, 13057–13062 (1997).

    Article  CAS  Google Scholar 

  12. Nimmakayalu, M., Henegariu, O., Ward, D.C. & Bray-Ward, P. Simple method for preparation of fluor/hapten-labeled dUTP. Biotechniques 28, 518–522 (2000).

    Article  CAS  Google Scholar 

  13. Stanford University School of Medicine, Brown lab. The MGuide. http://cmgm.stanford.edu/pbrown/mguide/index.html.

  14. National Human Genome Research Institute Division of Intramural Research. Microarray Project (uAP). http://www.nhgri.nih.gov/DIR/Microarray/main.html.

  15. The University of Iowa. BMAP Project. http://brainest.eng.uiowa.edu.

  16. Chen, Y., Dougherty, E.R. & Bittner, M.L. Ratio-based decisions and the quantitative analysis of cDNA microarray images. Biomedical Optics 2, 364–374 (1997).

    Article  CAS  Google Scholar 

  17. Chen, Y. et al. Ratio statistics of gene expression levels and applications to microarray data analysis. Bioinformatics, in press (2002).

    Google Scholar 

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Acknowledgements

This project was partly supported by federal funds from the National Cancer Institute, National Institutes of Health, under contract number NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

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Authors and Affiliations

  1. Laboratory of Genetics, National Institute of Mental Health, Bethesda, 20892, MD

    Charlie C. Xiang, Mei Chen, Quang N. Phan & Michael J. Brownstein

  2. Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, 20892, MD

    Yidong Chen

  3. SAIC Frederick, Frederick, 21702, MD

    Olga A. Kozhich & Jason M. Inman

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  1. Charlie C. Xiang
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Correspondence to Michael J. Brownstein.

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The US Department of Health and Human Services has applied to patent the labeling method described in this manuscript. The patent is entitled, “Amine modified random primers for microarray detection.”

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Xiang, C., Kozhich, O., Chen, M. et al. Amine-modified random primers to label probes for DNA microarrays. Nat Biotechnol 20, 738–742 (2002). https://doi.org/10.1038/nbt0702-738

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