Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency

Ales Tichopad, Anamarija Dzidic, Michael W. Pfaffl

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

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 β-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.

Original languageEnglish
Pages (from-to)2053-2056
Number of pages4
JournalBiotechnology Letters
Volume24
Issue number24
DOIs
StatePublished - Dec 2002

Keywords

  • Gene quantification
  • PCR efficiency
  • Primers
  • RT-PCR

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