qRAT: an R-based stand-alone application for relative expression analysis of RT-qPCR data

Abstract

Abstract Background Reverse transcription quantitative real-time PCR (RT-qPCR) is a well-established method for analysing gene expression. Most RT-qPCR experiments in the field of microbiology aim for the detection of transcriptional changes by relative quantification, which means the comparison of the expression level of a specific gene between different samples by the application of a calibration condition and internal reference genes. Due to the numerous data processing procedures and factors that can influence the final result, relative expression analysis and interpretation of RT-qPCR data are still not trivial and often necessitate the use of multiple separate software packages capable of performing specific functions. Results Here we present qRAT, a stand-alone desktop application based on R that automatically processes raw output data from any qPCR machine using well-established and state-of-the-art statistical and graphical techniques. The ability of qRAT to analyse RT-qPCR data was evaluated using two example datasets generated in our laboratory. The tool successfully completed the procedure in both cases, returning the expected results. The current implementation includes functionalities for parsing, filtering, normalizing and visualisation of relative RT-qPCR data, like the determination of the relative quantity and the fold change of differentially expressed genes as well as the correction of inter-plate variation for multiple-plate experiments. Conclusion qRAT provides a comprehensive, straightforward, and easy-to-use solution for the relative quantification of RT-qPCR data that requires no programming knowledge or additional software installation. All application features are available for free and without requiring a login or registration.

Keywords

• Reverse transcription quantitative real-time (RT-qPCR )
• Shiny
• Relative quantification
• Relative expression analysis
• Fold change
• R