Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package

Abstract

When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references—delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis.Key features• Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder.• Identification of endogenous references for normalization of RT-qPCR data.• Create publication-ready graphs and tables output.• Step-by-step guide dialog window for novice R users.Graphical overviewSimple workflow diagram. Two main workflow paths are presented. A) Using the RefSeeker wizard allows non-R programmers to easily load data and choose between selected output formats. B) Command line interface provides more options to control input and output formats and to automate analysis.