Shape based kinetic outlier detection in real-time PCR

Background: Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results: Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molec...

• Type: Journal
• Year: 2010
• Where: BMCBI
• Authors: Davide Sisti, Michele Guescini, Marco B. L. Rocchi, Pasquale Tibollo, Mario D'Atri, Vilberto Stocchi
• Keywords: BMCBI 2010, PCR Efficiency, Quantification, Real-time Pcr