A PCR primer bank for quantitative gene expression analysis
A PCR primer bank for quantitative gene expression analysis
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DOI：
10.1093/nar/gng154
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发表时间：
2003-12-15
影响因子：
14.9
通讯作者：
Seed, B
中科院分区：
文献类型：
--
作者：
Wang, XW;Seed, B
虽然微阵列分析的基因表达谱是评估全球转录活性水平的有用工具，但与数据集相关的可变性通常需要通过一些其他方法来验证观察到的差异，例如实时定量聚合酶链式反应(Real-time PCR)。然而，在后者中经常观察到非靶基因的非特异性扩增，这与不使用引物特异性标记时40%的实时聚合酶链式反应的分析结果相类似。在这里，我们提出了一种实验验证的算法，用于在年龄经济规模上识别转录本特异的PCR引物，该算法可以应用于具有序列无关检测方法的实时PCR。一个名为PrimerBank的在线数据库已经为研究人员创建，以检索他们感兴趣的基因的引物信息。PrimerBank目前包含147个404个引物，涵盖了大多数已知的人类和老鼠基因。对112对子集进行了常规和实时聚合酶链式反应(RT-PCR)检测，成功率为98.2%。
Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in similar to40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on age nomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.