Huber Martin, Wei Tai-Fen, Müller Uwe R, Lefebvre Phil A, Marla Sudhakar S, Bao Y Paul
Nanosphere Inc., 4088 Commercial Ave., Northbrook, IL 60062, USA.
Nucleic Acids Res. 2004 Oct 8;32(18):e137. doi: 10.1093/nar/gnh133.
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-dT20 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 microg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.
基于微阵列的基因表达分析在现代生物学中起着关键作用，并有望进入分子诊断领域。当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。转化为cRNA涉及一个靶标扩增步骤，该步骤克服了与常用荧光检测方法相关的低灵敏度问题。在此，我们提出了一种新型的基于微阵列的无酶基因表达系统，该系统使用未扩增的人总RNA样本作为靶核酸。通过用寡聚dT20修饰的金纳米颗粒探针靶向多聚A尾、通过自动金相术进行信号放大以及随后测量纳米颗粒介导的光散射来完成对微阵列结合的RNA分子的检测。纳米颗粒探针提供的高灵敏度允许在2小时杂交中从低至0.5微克未扩增的人总RNA中检测差异基因表达，而无需复杂的样品标记步骤。
