Nature Biotechnology
18, 457 - 459 (2000)
doi:10.1038/74546
High-fidelity mRNA amplification for gene profilingEna Wang1, 3, Lance D. Miller2, 3, Galen A. Ohnmacht1, Edison T. Liu2
& Francesco M. Marincola11
Surgery Branch, Division of Clinical Sciences, National Cancer Institute and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD
2
Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Gaithersburg, MD
3
These two authors contributed equally to this project.
Correspondence should be addressed to Francesco M. Marincola marincola@nih.govThe 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  g of total RNA (T-RNA) and 2−5 g 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.
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