BestKeeper

BestKeeper is a free, Excel-based software tool for the analysis of quantitative real-time PCR (qPCR) data, used to identify and validate stable reference genes for normalization.

Developed and described in 2004 by Pfaffl, Tichopad, Prgomet, and Neuhauser, BestKeeper analyzes Ct values across samples to assess candidate reference gene stability. Its core workflow computes descriptive statistics such as standard deviation (SD) and coefficient of variation (CV) for each candidate gene, constructs a BestKeeper index from the geometric mean of the candidate Ct values, and evaluates stability by pairwise correlations with the index and between genes. Genes with high SD or low correlation are flagged as unstable.

The software outputs stability metrics, correlation tables, and charts within an Excel workbook, enabling researchers to select one or more reference genes for normalization. BestKeeper emphasizes consistent PCR efficiency across genes and assumes stable expression of reference genes across samples.

Limitations and context: BestKeeper has been influential in shaping qPCR normalization but is often used alongside other methods, given concerns about its assumptions and sensitivity to outliers. It has been complemented by or contrasted with other approaches such as geNorm and NormFinder, which offer alternative strategies for selecting reference genes. Over time, while newer tools have emerged, BestKeeper remains cited as a foundational method and is still accessible in various repositories as a historical and practical option for qPCR data analysis.