Technical Report | Published:

High-fidelity mRNA amplification for gene profiling

Nature Biotechnologyvolume 18pages457459 (2000) | Download Citation

Subjects

Abstract

The completion of the Human Genome Project1 has made possible the comprehensive analysis of gene expression2,3, and cDNA microarrays are now being employed for expression analysis in cancer cell lines4 or excised surgical specimens5. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50–200 μg of total RNA (T-RNA) and 2–5 μg poly(A) RNA6. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal7,8,9 have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA10,11 or cDNA12 show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures11,13,14,15,16, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification10 with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Collins, F.S. et al. New goals for the U.S. Human Genome Project. Science 282, 682–629 (1998).

  2. 2

    Lander, E.S. The new genomics: global view of biology. Science 274, 536–539 (1996).

  3. 3

    Brown, P.O. & Botstein, D. Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21, 33–37 (1999).

  4. 4

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

  5. 5

    Perou, C.M. et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc. Natl. Acad. Sci. USA 96, 9212–9217 (1999).

  6. 6

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

  7. 7

    Chen, J.J. et al. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 51, 313–324 (1998).

  8. 8

    Rajeevan, M.S., Dimulescu, I.M., Unger, E.R. & Vernon, S.D. Chemiluminescent analysis of gene expression on high-density filter arrays. J. Histochem. Cytochem. 47, 337–342 (1999).

  9. 9

    Zejie, Y., Xiaoyi, W., Yu, T. & Huan, H. The method of micro-displacement measurement to improve the space resolution of array detector. Med. Engineer. Phys. 20, 149–151 (1998).

  10. 10

    Phillips, J. & Eberwine, J.H. Antisense RNA amplification: a linear amplification method for analyzing the mRNA population from single living cells. Methods 10, 283–288 (1996).

  11. 11

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

  12. 12

    Trenkle, T., Welsh, J., Jung, B., Mathieu-Daude, F. & McClelland, M. Non-stoichiometric reduced complexity probes for cDNA arrays. Nucleic Acids Res. 26, 3883–3891 (1998).

  13. 13

    Luo, L. et al. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat. Med. 5, 117–122 (1999).

  14. 14

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

  15. 15

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

  16. 16

    Kacharmina, J.E., Crino, P.B. & Eberwine, J.H. Preparation of cDNA from single cells and subcellular regions. Methods Enzymol. 303, 3–19 (1999).

  17. 17

    Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

  18. 18

    Matz, M. et al. Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558–1560 (1999).

  19. 19

    Mahadevappa, M. & Warrington, J.A. A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat. Biotechnol. 17, 1134–1136 (1999).

  20. 20

    Schena, M. et al. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA 93, 10614–10619 (1996).

  21. 21

    National Human Genome Research Institute. Division of Intramural Research. Microarray ProjectAP). http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/img_analysis.html.

Download references

Acknowledgements

The authors wish to acknowledge John Powell (CIT) for establishing the NCI microarray database (mAdb) and accessibility of analytical tools, and Louis Staudt (NCI) for array clones.

Author information

Author notes

  1. Ena Wang and Lance D. Miller: These two authors contributed equally to this project.

Affiliations

  1. Division of Clinical Sciences, National Cancer Institute and the Department of Transfusion Medicine, Surgery Branch, Clinical Center, National Institutes of Health, Bethesda, MD

    • Ena Wang
    • , Galen A. Ohnmacht
    •  & Francesco M. Marincola
  2. Division of Clinical Sciences, Medicine Branch, National Cancer Institute, National Institutes of Health, Gaithersburg, MD

    • Lance D. Miller
    •  & Edison T. Liu

Authors

  1. Search for Ena Wang in:

  2. Search for Lance D. Miller in:

  3. Search for Galen A. Ohnmacht in:

  4. Search for Edison T. Liu in:

  5. Search for Francesco M. Marincola in:

Corresponding author

Correspondence to Francesco M. Marincola.

About this article

Publication history

Received

Accepted

Issue Date

DOI

https://doi.org/10.1038/74546

Further reading