Abstract
BackgroundLinear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis.FindingsUtilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification.ConclusionsThe LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.
Highlights
Real-time qPCR has provided the foundation for a plethora of applications in basic research, biomedical diagnostics and pathogen detection [1,2,3]
The Linear regression of efficiency (LRE) Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification
Absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples
Summary
Real-time qPCR has provided the foundation for a plethora of applications in basic research, biomedical diagnostics and pathogen detection [1,2,3]. Foremost is the difficulty of implementing absolute quantification, due to the necessity of constructing target-specific standard curves [4]. This makes absolute quantification impractical for large-scale applications that require quantification of more than a handful of targets. Originating from the application of sigmoidal mathematics to model PCR amplification, linear regression of efficiency (LRE) provides an alternative approach to real-time qPCR, in which absolute quantification can be conducted without standard curves [5,6,7]. Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. A major challenge of implementing LRE quantification is the labor intensive nature of the analysis
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