referencegenes

Reference genes, often called housekeeping genes, are genes that are presumed to maintain stable expression across different samples and conditions. They serve as internal controls to normalize measurements of target gene expression, helping to correct for variation in RNA quantity and quality, reverse transcription efficiency, and other technical factors that can influence data.

Selecting an appropriate reference gene requires empirical validation for the specific experiment. Researchers typically evaluate several candidate genes for expression stability using statistical approaches and tools such as geNorm, NormFinder, BestKeeper, or the ΔCt method. A normalization factor is usually calculated as the geometric mean of the expression levels (or Ct values) of the chosen reference genes, and the number of genes used may be increased if stability is not sufficient.

In practice, reference genes are most commonly used in RT-qPCR experiments to normalize target gene expression. In RNA-Seq and other high-throughput platforms, normalization often relies on transcriptome-wide scaling factors, read counts, or spike-in controls; however, reference genes can still serve as useful benchmarks or supplementary normalization checks in some workflows. There is no universal reference gene; stability is context-dependent and can vary by tissue, developmental stage, treatment, or disease state.

Common reference genes include ACTB (beta-actin), GAPDH, 18S rRNA, B2M, RPLP0, HPRT1, PPIA, and TUBB, but their suitability must be validated for each study. Transparent reporting should state which genes were used as references and why, along with the validation approach used to establish their stability.