CAmpER - A software for the calculation of amplification efficiencies for real-time PCR-experiments

Quantitative real-time PCR has become one of the fundamental methods of modern molecular biology for the measurement of gene expression. For the analysis of a real-time PCR experiment it is crucial to have exact knowledge of the underlying PCR kinetics. Especially the amplification efficiencies of an experiment are of importance for the analysis of gene expression ratios. For this reason the software CAmpER was designed as a software-tool for the automatic analysis, annotation, and storage of real-time PCR experiments performed with different real-time PCR systems, currently the Lightcycler® (Roche Diagnostics) and the Opticon® (Bio-Rad Laboratories, Inc.).

CAmpER obtains the experiment data by automatically importing the fluorescence and melting-curve data from the output files of the real-time PCR-system. Based on the imported fluorescence data the amplification efficiencies are calculated for single samples by two different independent algorithms. The first algorithm is based on the DART-PCR method (Peirson et al., Nucleic Acid Research, 31(14):e73 2003), the second algorithm is based on a four parametric logistic model (Tichopad et al., Nucleic Acid Research, 31(20):e122 2003) of the fluorescence curve. Both algorithms were revised and improved to be applicable for different qPCR systems. The calculated amplification efficiencies are used to calculate efficiency-corrected crossing points. These corrected crossing points can be used to estimate gene expression ratios for all samples, which are more accurate than gene expression ratios calculated from uncorrected crossing points.

Another function of CAmpER is the consistent storage of all relevant information of a real-time PCR experiment, giving CAmpER a basic LIMS functionality. It allows users to annotate the samples used in the experiment as well as the experimental parameters in a standardized format. The annotation information and all other experiment data is stored using a relational database management system (MySQL). The modular design of CAmpER allows for extension of the software, for example to support additional real-time PCR systems in the future. To ensure convenient access to stored data and easy use of the implemented functions a convenient user interface was designed. This user interface is implemented as a web-based frontend, making a local installation of CAmpER needless.