Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA -: art. no. e137
Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA -: art. no. e137
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DOI：
10.1093/nar/gnh133
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Bao, YP
中科院分区：
文献类型：
--
作者：
Huber, M;Wei, TF;Bao, YP
基于微阵列的基因表达分析在现代生物学中发挥着关键作用，并有望进入分子诊断领域。目前基于微阵列的基因表达系统通常需要将 mRNA 酶促转化为标记的 cDNA 或 cRNA。转换为 cRNA 涉及目标扩增步骤，该步骤克服了常用荧光检测方法的低灵敏度。在此，我们提出了一种新型无酶、基于微阵列的基因表达系统，该系统使用未扩增的总人类 RNA 样本作为靶核酸。微阵列结合的 RNA 分子的检测是通过使用oligo-dT(20)修饰的金纳米颗粒探针靶向多聚腺苷酸尾部、通过自动金相学放大信号以及随后测量纳米颗粒介导的光散射来完成的。纳米颗粒探针提供的高灵敏度允许在 2 小时杂交中从少至 0.5 杯未扩增的总人类 RNA 中进行差异基因表达，而无需复杂的样品标记步骤。
Microarray-based gene expression analysis plays a pivotal role in modern biology and is poised to enter the field of molecular diagnostics. Current microarray-based gene expression systems typically require enzymatic conversion of mRNA into labeled cDNA or cRNA. Conversion to cRNA involves a target amplification step that overcomes the low sensitivity associated with commonly used fluorescent detection methods. Herein, we present a novel enzyme-free, microarray-based gene expression system that uses unamplified total human RNA sample as the target nucleic acid. The detection of microarray-bound RNA molecules is accomplished by targeting the poly-A tail with an oligo-dT(20) modified gold nanoparticle probe, signal amplification by autometallography, and subsequent measurement of nanoparticle-mediated light scattering. The high sensitivity afforded by the nanoparticle probes allows differential gene expression from as little as 0.5 mug unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.