Quantitative cDNA-AFLP analysis for genome-wide expression studies
Quantitative cDNA-AFLP analysis for genome-wide expression studies
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DOI：
10.1007/s00438-003-0830-6
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发表时间：
2003-05-01
影响因子：
3.1
通讯作者：
Zabeau, M
中科院分区：
文献类型：
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作者：
Breyne, P;Dreesen, R;Zabeau, M
建立了一种用于全基因组表达分析的cDNA-AFLP方法。我们证明，这种方法是一种有效的工具，定量转录谱和一个有效的替代芯片。通过一系列选择性PCR扩增筛选由限制性内切酶逆转录的信使RNA产生的独特转录物标签。基于计算机模拟分析，选择确保所有mRNA中至少60%由信息序列标签表示的酶组合。该方法的灵敏度和特异性允许人们检测低表达基因并区分同源序列。准确的基因表达谱的条带强度的定量分析，并揭示了转录活性的细微差异。对烟草中细胞周期调控基因的详细筛选证明了该技术用于全基因组表达分析的有用性。
An improved cDNA-AFLP method for genome-wide expression analysis has been developed. We demonstrate that this method is an efficient tool for quantitative transcript profiling and a valid alternative to microarrays. Unique transcript tags, generated from reverse-transcribed messenger RNA by restriction enzymes, were screened through a series of selective PCR amplifications. Based on in silico analysis, an enzyme combination was chosen that ensures that at least 60% of all the mRNAs were represented by an informative sequence tag. The sensitivity and specificity of the method allows one to detect poorly expressed genes and distinguish between homologous sequences. Accurate gene expression profiles were determined by quantitative analysis of band intensities, and subtle differences in transcriptional activity were revealed. A detailed screen for cell cycle-modulated genes in tobacco demonstrates the usefulness of the technology for genome-wide expression analysis.