High-fidelity mRNA amplification for gene profiling

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.

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Figure 1: (A) Assessment of random or labeling bias by hybridization of differentially labeled aRNA target (3 μg) amplified from the same melanoma cell line to 2,008-gene OncoChip.
Figure 2: (A) Grading of outlier reproducibility in mRNA, T-RNA, and aRNA hybridizations.
Figure 3: (A) Low-stringency cluster diagram of reproducible and anomalous (discordant) outliers.

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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.

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Correspondence to Francesco M. Marincola.

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Wang, E., Miller, L., Ohnmacht, G. et al. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18, 457–459 (2000). https://doi.org/10.1038/74546

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