A high-performance, small-scale microarray for expression profiling of many samples in Arabidopsis-pathogen studies
A high-performance, small-scale microarray for expression profiling of many samples in Arabidopsis-pathogen studies
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DOI：
10.1111/j.1365-313x.2006.02972.x
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发表时间：
2007-02-01
期刊：
影响因子：
7.2
通讯作者：
Katagiri, Fumiaki
中科院分区：
文献类型：
--
作者：
Sato, Masanao;Mitra, Raka M.;Katagiri, Fumiaki
对生物系统行为的研究通常需要监测大量样本中许多基因的表达。虽然全基因组阵列提供了高质量的基因表达谱，但其高昂的成本通常限制了可以研究的样本数量。虽然廉价的代表感兴趣基因的小规模阵列可以用于许多应用，但用传统的小规模微阵列获得准确的测量是具有挑战性的。我们已经开发了一种小规模的微阵列系统，可以产生高度准确和可重复性的表达测量结果。这是通过实施稳定的基于基因的分位数归一化方法进行阵列到阵列归一化，以及允许使用统计模型校正打印尖端和不均匀杂交的影响的探针打印设计来实现的。该数组测量单个样本中的表达式值，而不是两个样本之间的比率。这允许在许多样本之间进行准确的比较。该阵列通常在技术上复制的样本之间产生高于0.99的相关系数。从该阵列和Affymetrix基因芯片确定的表达比率之间的相关系数为0.88，通过定量RT-PCR和将已知数量的特定RNA添加到用于简档分析的RNA样本中，证明了准确性。用该阵列比较野生型、rps2和ndr1突变株对一株表达avrRpt2的丁香假单胞菌的侵染反应。这些结果表明，NDR1除了影响RPS2依赖的防御信号通路外，还影响一条防御信号通路(S)，并表明微阵列是系统分析拟南芥疾病信号网络的有力工具。
Studies of the behavior of biological systems often require monitoring of the expression of many genes in a large number of samples. While whole-genome arrays provide high-quality gene-expression profiles, their high cost generally limits the number of samples that can be studied. Although inexpensive small-scale arrays representing genes of interest could be used for many applications, it is challenging to obtain accurate measurements with conventional small-scale microarrays. We have developed a small-scale microarray system that yields highly accurate and reproducible expression measurements. This was achieved by implementing a stable gene-based quantile normalization method for array-to-array normalization, and a probe-printing design that allows use of a statistical model to correct for effects of print tips and uneven hybridization. The array measures expression values in a single sample, rather than ratios between two samples. This allows accurate comparisons among many samples. The array typically yielded correlation coefficients higher than 0.99 between technically duplicated samples. Accuracy was demonstrated by a correlation coefficient of 0.88 between expression ratios determined from this array and an Affymetrix GeneChip, by quantitative RT-PCR, and by spiking known amounts of specific RNAs into the RNA samples used for profiling. The array was used to compare the responses of wild-type, rps2 and ndr1 mutant plants to infection by a Pseudomonas syringae strain expressing avrRpt2. The results suggest that ndr1 affects a defense-signaling pathway(s) in addition to the RPS2-dependent pathway, and indicate that the microarray is a powerful tool for systems analyses of the Arabidopsis disease-signaling network.