An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays
An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays
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DOI：
10.1186/1471-2164-6-66
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发表时间：
2005-05-08
期刊：
影响因子：
4.4
通讯作者：
Wu, WS
中科院分区：
文献类型：
--
作者：
Gadgil, M;Lian, W;Wu, WS
背景：DNA 微阵列是基因表达探索的宝贵工具。在双染料微阵列中，杂交后比较两个样品的荧光强度，每个样品用不同的染料标记。为了比较大量样品，广泛使用“参考设计”，其中所有 RNA 样品都与一个共同的参考杂交。基因组 DNA 是用作通用参考的有吸引力的候选者，特别是对于非编码序列百分比较低的细菌系统。然而，由有义链和反义链组成的基因组 DNA 与微阵列杂交中通常使用的单链 cDNA 不同。 “参考”中反义链的存在会导致溶液中互补标记链之间发生反应，并可能导致测定结果偏离真实值。结果：我们开发了一个数学模型来预测在微阵列测定中使用基因组 DNA 作为参考的有效性。该模型预测该测定可以准确估计各种初始 cDNA 浓度的相对浓度。使用基因组 DNA 作为参考的 DNA 微阵列测定的实验结果与通过两个 cDNA 样品之间直接杂交获得的结果相关良好。该模型预测标记基因组 DNA 链和固定链的初始浓度以及杂交时间不会显着影响测定性能。当固定链和移动链之间的杂交速率常数较低时，测定性能随杂交时间和初始 cDNA 浓度而变化。对于使用固定单链微阵列的情况，使用基因组 DNA 作为参考的杂交结果将与所有条件下的真实比率相对应。结论：使用数学模型进行的模拟以及此处介绍的实验研究显示了使用基因组 DNA 作为参考的微阵列测定的潜在效用。我们的结论是，使用基因组 DNA 作为参考 DNA 应极大地促进比较转录组分析。
Background: DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values.Results: We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions.Conclusion: Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.