Abstract
BackgroundReal-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.ResultsA systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). 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 SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 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. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 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 ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.ConclusionNone 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.
Highlights
Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation
Transcription profiling of soybean reference genes A RT-qPCR assay based on SYBR Green detection was carried out to examine the stability of the expression of the 14 candidate genes (Table 1)
The expression level of the candidate reference genes are presented as quantification cycle (Cq) values (Figure 1)
Summary
Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Used reference genes include ribosomal RNA (18SrRNA) and a number of housekeeping genes, such as those encoding actin (ACT), tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), polyubiquitin (UBQ) and elongation factor 1-α (EF1α) [1,6,9,10]. These genes have been assumed to be constitutively expressed, as they are involved in basic and ubiquitous cellular processes [1,5,9,11]. A single reference gene is inadequate, and any such reliance is likely to produce erroneous conclusions vis-à-vis expression patterns [15,16,17,18]
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