Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification
Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification
复制标题
DOI：
10.2144/04374st02
复制
发表时间：
2004-10-01
期刊：
影响因子：
2.7
通讯作者：
Thorgeirsson, SS
中科院分区：
文献类型：
--
作者：
Kaposi-Novak, P;Lee, JS;Thorgeirsson, SS
基于单链长寡核苷酸(50-70-mer)的微阵列比传统的cDNA微阵列有几个优点。这些优点包括易于制备探头、低成本的阵列生产、以及在处理探头过程中的低交叉污染。然而，利用传统的基于寡核苷酸(DT)-T7启动子的扩增，利用寡核苷酸微阵列中寡核苷酸探针的单链性质(正义链)，利用少量总RNA来分析全球基因表达的应用受到阻碍。在这份报告中，我们描述了产生反义标记的cDNA靶的改进的RNA扩增方法，并成功地应用于寡核苷酸微阵列基因表达分析。在第一轮实验中，用寡核苷酸(DT)(24)T7启动的逆转录和T7RNA聚合酶体外转录线性扩增mR NA。在第二轮实验中，用随机9聚体T3引物和T3RNA聚合酶扩增正义链扩增RNA(ArNA)。在Arna前产生荧光标记的CDAA靶标并与寡核苷酸微阵列杂交。我们的数据显示，扩增提供了高度重复性的结果，扩增的样品和未扩增的样品之间的显著相关性证明了这一点。我们还证明，来自激光显微解剖的肿瘤样本的RNA的扩增复制了从相同样本的总RNA中获得的基因表达谱。
Single-stranded long oligonucleotide-based (50- to 70-mer) Microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array production, and low cross-contamination during probe handling. However the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotiole microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)(24)T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDAA targets were generated front the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.