Identification of Four Soybean Reference Genes for Gene Expression Normalization

Gene expression analysis requires the use of reference genes constitutively expressed independently of tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin, and elongation factor 1‐α) are commonly used as reference genes with the assumption that they are uniformly expressed. In many cases, however, this assumption was shown to be incorrect. To provide reliable reference genes in soybean [ Glycine max (L.)], we surveyed a set of genes that showed little variation in a nodulation study across a series of soybean microarray experiments. More than 200 putative reference genes were identified. We focused on 18 for further analysis using additional cDNA and Affymetrix arrays and quantitative reverse‐transcription polymerase chain reactions. Taken together, these experiments allowed us to test the expression stability of these genes in 130 different conditions, confirming four soybean genes as new reference genes (annotated as ATP‐binding cassette [ABC] transporter, F‐box protein family, metalloprotease, and CDPK‐related protein kinase). These genes should be useful for normalization of gene expression studies in soybean, an important crop plant.