Abstract
Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships1,2. However, the available procedures require multiple steps, are labor intensive and time consuming3, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably2,3,4,5. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription–PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 × 1011-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.
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19 August 2002
note on SGML, appended PDF, print will be correct (as will final issue PDF)
Notes
*Note: Due to a proofreading error by the authors in the AOP version of the paper, one of the author's names, Carolyn Modi, was spelled incorrectly. This mistake has been corrected in the HTML version and will appear correctly in print. The PDF version available online has been appended.
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Acknowledgements
We thank D. Nallainathan for technical assistance, P. Macgregor and M. Albert for their assistance with the linear amplification protocols, C. Holness and colleagues at SyStemix Inc. for DNA sequencing, and P. Subarsky for helpful discussion and advice. This work was supported by an operating grant to N.N.I. from the National Cancer Institute of Canada, and to N.W. from the Ontario Research and Development Challenge Fund.
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Iscove, N., Barbara, M., Gu, M. et al. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat Biotechnol 20, 940–943 (2002). https://doi.org/10.1038/nbt729
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DOI: https://doi.org/10.1038/nbt729
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