Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency
Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency
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DOI：
10.1023/a:1021319421153
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发表时间：
2002-12-01
影响因子：
2.7
通讯作者：
Pfaffl, MW
中科院分区：
文献类型：
--
作者：
Tichopad, A;Dzidic, A;Pfaffl, MW
测试了引物选择对实时聚合酶链反应（RT-PCR）性能的影响。利用不同长度的引物扩增β -肌动蛋白序列。长度的差异导致RT-PCR性能的差异。采用四参数s型模型对实时数据进行数学近似，研究了PCR的动力学参数。该模型描述了放大轨迹的完整动力学。对该模型产生的参数进行统计研究表明，由性能良好的引物引发的放大效率较高的反应具有较低的可变性，因此更适合于测量目的。
The effect of primer selection on real-time polymerase chain reaction (RT-PCR) performance was tested. Primer sets of varying length of product were used to amplify the sequence of beta-actin. Variability in length caused variability in RT-PCR performance. Kinetic parameters of PCR were studied by mathematical approximation of real-time data by means of a four-parametric sigmoid model. This model describes the full kinetics of the amplification trajectory. Statistical exploration of parameters yielded by this model revealed that reactions with higher amplification efficiency - primed by well-performing primers - proceed with lower variability and are therefore better suited for measurement purposes.