NormFinder

NormFinder is a statistical algorithm and software tool used to identify stably expressed reference genes for normalization in real-time quantitative PCR (qPCR) and related gene-expression analyses. It was introduced by Andersen CL, Jensen JL, and Ørntoft TF in 2004 as a model-based approach to selecting candidate normalization genes.

The method employs a linear model to estimate intra- and inter-group variation of candidate reference genes across samples. For each gene, NormFinder computes a stability value, with lower values indicating more stable expression suitable for normalization. In addition to ranking individual genes, the algorithm can identify the best combination of two genes to form a normalization factor that minimizes overall variation across the study design, particularly when group structure is present (for example, cases vs. controls).

Usage typically involves providing expression data for a set of candidate reference genes across samples, along with grouping information and any known experimental conditions. The output includes a ranked list of genes by stability and recommendations for normalization strategies, including which single gene or pair to use. NormFinder helps avoid relying on a single unstable gene and can account for systematic differences between subgroups.

NormFinder has been implemented in several software packages and is widely cited in the literature as a robust method for reference-gene selection. It is often used in conjunction with other normalization tools, such as geNorm or BestKeeper, to assess and validate suitable housekeeping genes. Limitations include dependence on the initial candidate gene set and appropriate experimental design; results improve with careful selection of candidate references and explicit group definitions.