In silico tools for qPCR assay design and data analysis.
qPCR instruments are supplied with basic software packages that enable the measurement of fluorescent changes, calculations of quantification cycle (Cq) values, the generation of standard curves and subsequent relative target nucleic acid quantity determination. However, detailed assessments of the technical parameters underlying Cq values and their translation into biological meaningful results require validation of these basic calculations through further analyses such as qPCR efficiency correction, normalization to multiple reference genes, averaging and statistical tests. Some instruments incorporate some of these features, while others offer additional tools to complement the basic running software, in many cases providing those that are described below. In this chapter, there is a detailed description of some of these programs and recommended strategies for the design of robust qPCR assays. Some of the packages available for validation of the resulting Cq data and detailed statistical analysis are described.