A novel, high-performance random array platform for quantitative gene expression profiling
A novel, high-performance random array platform for quantitative gene expression profiling
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DOI：
10.1101/gr.2739104
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发表时间：
2004-11-01
期刊：
影响因子：
7
通讯作者：
Chee, MS
中科院分区：
文献类型：
--
作者：
Kuhn, K;Baker, SC;Chee, MS
我们开发了一种新的微阵列技术，用于在基于随机组装的微珠阵列基础上进行定量基因表达谱分析。每个微珠携带一种基因特异性的探针序列。在一个阵列中，每种序列特异性微珠都有多个拷贝，这有助于提高测量的准确性和可靠性。我们对该系统进行了优化，以用于对哺乳动物RNA进行特异性和灵敏性分析，并且利用已知浓度的RNA对照，获得了对系统性能的如下评估：在哺乳动物多聚（A +）mRNA中的特异性为1 : 250,000；检测限为0.13皮摩尔；动态范围为3.2个对数级；在动态范围内有足够的精度以95%的置信度检测到1.3倍的差异。通过与人脑和肝脏之间表达差异的测量结果与定量实时聚合酶链反应（对于对数转换后的比率，R² = 0.98，对于20个基因，最佳拟合线的斜率 = 1.04）的一致性进行了验证。使用小鼠B细胞和T细胞模型系统进一步验证了定量性能。我们发现，在我们的系统中，59个在B细胞和T细胞之间显示出最大差异表达的基因中有42个有已发表的关于B细胞或T细胞特异性表达的报告。所有文献中的观察结果都与我们的结果一致。我们的实验是在一个96阵列矩阵系统上进行的，该系统仅需要100纳克的输入RNA，并使用标准微量滴定板来并行处理样品。我们的技术在分析多个样品方面具有优势，可扩展到基因组中所有已知基因，并且具有灵活性，允许在一个阵列中使用标准或定制的探针。
We have developed a new microarray technology for quantitative gene-expression profiling on the basis of randomly assembled arrays of beads. Each bead carries a gene-specific probe sequence. There are multiple copies of each sequence-specific bead in an array, which contributes to measurement precision and reliability. We optimized the system for specific and sensitive analysis of mammalian RNA, and using RNA controls of defined concentration, obtained the following estimates of system performance: specificity of 1:250,000 in mammalian poly(A(+)) mRNA; limit of detection 0.13 pM; dynamic range 3.2 logs; and Sufficient precision to detect 1.3-fold differences with 95% confidence within the dynamic range. Measurements of expression differences between human brain and liver were validated by concordance with quantitative real-time PCR (R-2 = 0.98 for log-transformed ratios, and slope of the best-fit line = 1.04, for 20 genes). Quantitative performance was further verified using a mouse B- and T-cell model system. We found published reports of B- or T-cell-specific expression for 42 of 59 genes that showed the greatest differential expression between B- and T-cells in our system. All of the literature observations were concordant with our results. Our experiments were carried out on a 96-array matrix system that requires only 100 ng of input RNA and uses standard microtiter plates to process samples in parallel. Our technology has advantages for analyzing multiple samples, is scalable to all known genes in a genome, and is flexible, allowing the use of standard or custom probes in an array.