Wang E, Miller L D, Ohnmacht G A, Liu E T, Marincola F M
Surgery Branch, Division of Clinical Sciences, National Cancer Institute and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.
Nat Biotechnol. 2000 Apr;18(4):457-9. doi: 10.1038/74546.
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.
人类基因组计划的完成使得对基因表达进行全面分析成为可能，如今cDNA微阵列正被用于癌细胞系或手术切除标本的表达分析。然而，cDNA微阵列更广泛的应用受到所需RNA量的限制：总RNA（T-RNA）需要50 - 200微克，聚腺苷酸RNA（poly(A) RNA）需要2 - 5微克。为了拓宽cDNA微阵列的用途，一些旨在增强荧光信号的方法取得了一定程度的改进。致力于扩增起始poly(A) RNA或cDNA的方法显示出前景，因为检测灵敏度可以提高几个数量级。然而，尽管这些扩增程序被广泛使用，但尚未有对其局限性和偏差的系统评估记录。我们设计了一种程序，通过将反义RNA（aRNA）扩增与模板转换效应（Clonetech，加利福尼亚州帕洛阿尔托）相结合，优化低丰度RNA样本的扩增。从常用输入RNA的1:10,000到1:100,000扩增得到的aRNA的保真度与基于传统poly(A) RNA或T-RNA的阵列所观察到的表达谱相当。
