Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (<jats:italic>ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b</jats:italic>) and seven new candidates (<jats:italic>SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2</jats:italic>). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of <jats:italic>SKIP16, UKN1</jats:italic> and <jats:italic>UKN2</jats:italic> was overall the most stable. A combination of <jats:italic>ACT11, UKN1</jats:italic> and <jats:italic>UKN2</jats:italic> would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. <jats:italic>ACT11, TUA5</jats:italic> and <jats:italic>TIP41</jats:italic> were the most stably expressed when the photoperiod was altered, and <jats:italic>TIP41, UKN1</jats:italic> and <jats:italic>UKN2</jats:italic> when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with <jats:italic>ACT11, UKN2</jats:italic> and <jats:italic>TUB4</jats:italic> being the most stable genes. The relative gene expression level of <jats:italic>GmFTL3</jats:italic>, an ortholog of Arabidopsis <jats:italic>FT</jats:italic> (<jats:italic>FLOWERING LOCUS T</jats:italic>) was detected to validate the reference genes selected in this study.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.</jats:p> </jats:sec>