th.cyc calculates the number of cycles at which the fluorescence exceeds a defined threshold, called the threshold cycle (Ct). According to the MIQE guidelines the Ct is referred to as quantification cycle (Cq). The calculated Cq is a relative value, which depends on the template copy number, instrument, reagents, amplification efficiency and probe technology. Low Cqs correlate with high quantities template copy numbers. Real-time technologies enable the quantification of nucleic acids by calculation of specific curve parameters like the quantification point (Cq) and the amplification efficiency (AE) based on the kinetics of the amplification curve. The Cq represents the number of cycles (time for qIA) needed to reach a defined fluorescence signal level in the exponential phase of the amplification curve. The Cq can be determined from a fixed threshold value or by various analytical algorithm as described elsewhere (Bustin et al. 2009, Ruijter et al. 2013, Tellinghuisen et al. 2014).
amptester.gui is a graphical user interface for the amptester function. This function can be used for a fast and convenient analysis of amplification curve data. In addition it is possible to analyze the Cq (quantification cycle) and to perform a report generation of the analyzed data.
normalizer normalizes any data set using a chosen method (see Details). It may be used when the data from an experiment have considerable variation regarding the background and plateau signal.
Smoother is a wrapper for several smoothing functions including LOWESS, Moving Average, Friedman's SuperSmoother, Cubic Spline and Savitzky-Golay smoothing filter, Friedman's SuperSmoother, and Whittaker smoother for amplification curve data.
A function inder ("in" + "der" = interpolate derivatives) for interpolating first and second derivatives using the five-point stencil. Therefore this function can be used to estimate the Cq (cycle of quantification) of an amplification curve. First positive derivative also known as First Derivative Maximum (FDM) and the Second Derivative Maximum (SDM) are calculated this way (Ruijter et al. 2013). However, from the mathematical point of view it can also be used to calculate the melting point for melting curve analysis (compare Roediger et al. 2013) provided that the sign of the derivative is changed.
A quantitative PCR (qPCR) with the DNA binding dye (EvaGreen) (Mao et al. 2007) was performed in a Bio-Rad iQ5 thermo cycler. The cycle-dependent increase of the fluorescence was quantified at the annealing step (59.5 degrees Celsius).
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