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

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is crucial for RNA expression analysis.
• Accurate normalization of RT-qPCR data relies on identifying stable endogenous reference genes.
• Existing tools like RefFinder offer valuable analysis but have cumbersome interfaces.

Purpose of the Study:

• To develop an R package, RefSeeker, for efficient and user-friendly RefFinder analysis.
• To facilitate the identification of stable endogenous references for RT-qPCR data normalization.
• To provide tools for creating publication-ready graphs and tables from stability analysis.

Main Methods:

• Development of the RefSeeker R package.
• Integration of five stability assessment algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder.
• Implementation of raw data import and data output functionalities for graphs and tables.
• Inclusion of a step-by-step guide dialog window for novice R users.

Main Results:

• RefSeeker enables fast and straightforward stability analysis of candidate endogenous references.
• The package supports data normalization for both coding (mRNA) and non-coding (miRNA) RNA expression studies.
• Publication-ready outputs (graphs and tables) are generated, enhancing data presentation.

Conclusions:

• RefSeeker significantly improves the efficiency and ease of selecting reference genes for RT-qPCR.
• This R package is a valuable tool for researchers conducting RNA expression analysis.
• RefSeeker democratizes advanced reference gene validation for users with varying R proficiency.