Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays
Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays
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DOI：
10.2144/01315dd03
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发表时间：
2001-11-01
期刊：
影响因子：
2.7
通讯作者：
Reynolds, MA
中科院分区：
文献类型：
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作者：
Evertsz, EM;Au-Young, J;Reynolds, MA
玻璃基因微阵列可以用来在一次实验中分析数千个基因靶点的表达情况。然而，在解释结果时，需要考虑杂交交叉反应的可能性。在这里，我们描述了一个代表四个不同功能类别(家族)的模型阵列的杂交实验：趋化因子，细胞色素P-450同工酶。G蛋白和蛋白酶。根据同源性评分算法(LALIGN)，为该阵列选择的cDNA克隆显示出从55%到100%的成对序列同源性。通过聚合酶链式反应扩增微阵列的靶点，并以4倍重复进行斑点以进行信号平均。通过聚合酶链式反应进一步扩增每个家族的一个指定靶点，以加入T7启动子序列，以生产合成RNA转录本。这些转录本被用来在不同的输入浓度下通过逆转录产生荧光杂交探针。正如预期的那样，杂交信号在匹配的靶分子上最高。相对于杂交探针序列包含的序列同源性小于80%的靶显示出0.6%至12%的交叉反应性。同一性大于80%的靶标显示出更高的交叉反应性(26%-57%)。对这些交叉反应信号与LALIGN确定的序列重叠长度、序列同源性百分比和同源性分数进行统计相关性分析。总体而言，序列同源性百分比是杂交交叉反应的最佳预测因子。这些结果为解释玻璃基因芯片数据提供了有用的指导。
Glass cDNA microarrays can be used to profile the expression of thousands of gene targets in a single experiment. However, the potential for hybridization cross-reactivity needs to be considered when interpreting the results. Here, we describe hybridization experiments with a model array representing four distinct functional classes (families): chemokines, cytochrome P-450 isozymes. G proteins, and proteases. The cDNA clones selected for this array exhibited pairwise sequence identities ranging from 55% to 100%, as determined by a homology scoring algorithm (LALIGN). Targets for microarraying were amplified by PCR and spotted in 4-fold replication for signal averaging. One designated target from each family was further amplified by PCR to incorporate a T7 promoter sequence for the production of synthetic RNA transcripts. These transcripts were used to generate fluorescent hybridization probes by reverse transcription at varying input concentrations. As expected, hybridization signals were highest at the matching target elements. Targets containing less than 80% sequence identity relative to the hybridization probe sequences showed cross-reactivities ranging from 0.6% to 12%. Targets containing greater than 80% identity showed higher cross-reactivities (26%-57%). These cross-reactive signals were analyzed for statistical correlation with the length of sequence overlap, percent sequence identity, and homology score determined by LALIGN. Overall, percent sequence identity was the best predictor of hybridization cross-reactivity. These results provide useful guidelines for interpreting glass cDNA microarray data.