High-fidelity mRNA amplification for gene profiling.
The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, 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) amplification 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.
Wang
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E
Surgery Branch, Division of Clinical Sciences, National Cancer Institute and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.
Miller
L D
LD
Ohnmacht
G A
GA
Liu
E T
ET
Marincola
F M
FM
D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D015470:Leukemia, Myeloid, Acute; D008545:Melanoma; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D016372:RNA, Antisense; D012333:RNA, Messenger