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A high-density probe array sample preparation method using 10- to 100-fold fewer cells

Nature Biotechnology volume 17, pages 1134–1136 (1999)Cite this article

One of the challenges of measuring gene expression is obtaining quantities of a homogeneous cell population sufficient for detecting low-abundance messages. Standard array protocols for monitoring gene expression typically require isolating poly(A) RNA from milligrams of tissue or millions of cells1,2. It is often hard to obtain a homogeneous clinical sample of this size, especially when assaying tissues that are difficult to harvest in large quantities, such as neurological tissue, small tumors, and cells obtained by needle biopsy3,4. A similar challenge arises in studying primary cells that are available in low quantities from in vivo sources such as the α, β, and δ cells of the islets of Langerhans in pancreas5,6.

Gene expression monitoring methods that enable transcript measurement from very few cells, such as reverse transcriptase (RT) PCR, have the disadvantage of measuring only one or a few messages simultaneously7. Furthermore, PCR amplification can skew the relative abundance levels of the message by selective amplification. Most non-array-based gene expression methods, such as in situ hybridization, northern blot, nuclease protection, and serial analysis of gene expression (SAGE), either require large amounts of starting material, are labor intensive and time consuming, or are not as readily scaleable and automatable as high-density oligonucleotide probe arrays7,8,9,10,11.

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Figure 1: Sample preparation method begins with total RNA extraction from cells or tissues.
Figure 2: (A) Number and expression level of transcripts detected in duplicate samples prepared by poly(A) and total RNA methods.
Figure 3: Images of hybridizations of 2.5, 5.0, 10.0, and 20.0 μg of labeled target from total RNA.

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Acknowledgements

We thank D. Lockhart, S. Dee, and N. Shah for helpful discussions during the course of this work. We thank S.Carter and A. Lau for aid in manuscript preparation. GeneChip is a registered trademark of Affymetrix, Inc.

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Authors and Affiliations

  1. research associate, Affymetrix, Inc., 3380 Central Expressway, Santa Clara, 95051, CA

    Mamatha Mahadevappa

  2. director of high-throughput screening, Affymetrix, Inc., 3380 Central Expressway, Santa Clara, 95051, CA

    Janet A. Warrington

Authors
  1. Mamatha Mahadevappa
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  2. Janet A. Warrington
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Mahadevappa, M., Warrington, J. A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat Biotechnol 17, 1134–1136 (1999). https://doi.org/10.1038/15124

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