Conditions to ensure competitive hybridization in two-color microarray: A theoretical and experimental analysis
Conditions to ensure competitive hybridization in two-color microarray: A theoretical and experimental analysis
复制标题
DOI：
10.2144/02326mt05
复制
发表时间：
2002-06-01
期刊：
影响因子：
2.7
通讯作者：
Ghosh, S
中科院分区：
文献类型：
--
作者：
Wang, YM;Wang, XJ;Ghosh, S
我们推导了一个理论模型，解释了文献中报道的双色微阵列杂交实验中观察到的某些偏差。我们表明，只有当两个差异标记的探针的杂交动力学相同时，才能实现真正的竞争。如果两个不同标记的探针的杂交动力学不同，这可能发生在两个探针的标记和杂交条件不同时，则观察到的差异表达成为靶标数量(即载玻片上的DNA斑点)的函数。我们使用这个模型来验证微阵列方法，通过确定四个选定的拟南芥基因和两个人类基因(β-肌动蛋白和GAPDH)的差异表达作为目标阵列数量的函数。我们通过建模和实验证明，在我们的专家的指导下，Cy5和Cy3标记的探针的速率常数是相同的。精神状况。因此。目标浓度不需要大大超过探针浓度。从所提供的数据可以明显看出，简单地处理单个杂交比率的计算并不能完全描述当今复杂的多物种实验中发生的事情。该验证方法易于实现，保证了双色微阵列数据的可靠性。
We derived a theoretical model that explains certain biases observed in the two-color microarray hybridization experiments reported in the literature. We show that true competition is achieved only when the hybridization kinetics of the two differentially labeled probes are the same. If the hybridization kinetics of the two differentially labeled probes is different, which can occur when the labeling and hybridization conditions for the two probes are dissimilar, then differential expression observed becomes a function of the amount of the target (i.e., DNA spotted on the slide). We use this model to validate the microarray methodology by determining the differential expression of four select Arabidopsis genes and two human genes (beta-actin and GAPDH) as a function of the amount of target arrayed. We show through both modeling and experiments that the rate constants for Cy5- and Cy3-labeled probes are the same under our expert. mental conditions. Therefore. the target concentrations need not greatly exceed the probe concentration. It is obvious from the data presented that a simple treatment of an individual hybridization rate calculation does not fully describe what is occuring in today's complex, multispecies experiments. The method of validation is easily implemented to ensure data reliability by two-color microarray.