OnlinePCR uses three methods for analysis of uploaded measurement file, one qualitative analysis and two quantitative methods: Standard curve and MAK2.
Standard curve method is a typical method for initial sample concentration estimation; it is commonly used in present-day qPCR analysis software. This method is based on construction of a standard curve from standard samples.
At first amplification curve is constructed for each sample by interpolation of its data (relative fluorescence values) measured during each PCR cycle. Consequently, a threshold value is calculated and used to obtain CT values (or crossing points) from standard samples. CT is a real number that represents a number of cycle in which fluorescence intesity of a particular standard sample outmatched the threshold. In other words, CT is a x-coordinate of point in which amplification curve of the standard intersects the level of threshold.
Next an array of points is created by putting log initial concentrations of standards and corresponding CT values together. These points are fitted using linear least squares in order to estimate a line - standard curve. Finally initial concentrations of unknown samples are calculated by evaluation of standard curve in their CT values.
Threshold value is automatically calculated by OnlinePCR using an iterative method by correlation coeficient optimization.
MAK2 stands for Mass Action Kinetic Method with 2 parameters. This method is based on non-linear regression of exponential phase of the amplification curve. Theoretical regression function formulation was based on simplification of the PCR kinetics model. MAK2 requires only one standard sample to be present among analyses samples.
Than all be will be taken to account in order to make the prediction more accurate.
Try these methods in OnlinePCR now. Its easy and fast. At first upload your measurement. Then just choose a method and channel and you'll see the results.
Of course, you can login to our demo application and browse through analysis results in read only mode.
Currently we recommend a standard curve method in OnlinePCR. We've tested it on large amount of data and received the most stable results. However, you should give a try to MAK2 method too.
This methods allows to perform quantitative analyses of samples with just a one standard sample. No preconfigured standard curve is required.
According to paper from Ruijter et al. (Methods 59, 2013, 32-46) MAK2 assumes constant amplification efficiency during the PCR reaction. However, theoretical analysis of polymerase chain reaction, from which MAK2 was derived, has revealed that amplification efficiency is not constant constant throughout PCR. While MAK2 quantification provides reliable estimates of target DNA concentration in a sample under normal qPCR conditions, MAK2 does not reliably quantify target concentration for qPCR assays with competimeters.
The method was developed in 2010 by Boogy and Woolf. For the purpose of OnlinePCR was method implemented in C++ by Rost and Španihel who utilized Zaharie's differential evolution for stable MAK 2 model regression.