BACKGROUND Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean. RESULTS All ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was ELF1B, CYP2 > ACT11 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different tissues under the same developmental stage, the rank was ELF1B, CYP2 > ACT2/7 > UBC2 > TUA > ELF1A > ACT11 > TUB > G6PD > UBQ10. For the developmental stage series, the stability rank was ACT2/7, TUA > ELF1A > UBC2 > ELF1B > TUB > CYP2 > ACT11 > G6PD > UBQ10. For photoperiodic treatments, the rank was ACT11, ELF1B > CYP2 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different times of the day, the rank was ELF1A, TUA > ELF1B > G6PD > CYP2 > ACT11 > ACT2/7 > TUB > UBC2 > UBQ10. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. Delta Ct approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from Delta Ct approach and geNorm indicated that ELF1B and CYP2 were the most stable HKGs, and UBQ10 and G6PD the most variable ones. Results from 'Stability index' analysis were different, with ACT11 and CYP2 being the most stable HKGs, and ELF1A and TUA the most variable ones. CONCLUSION Our data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and Delta Ct analysis, ELF1B and CYP2 could be used as internal controls to normalize gene expression in soybean, while UBQ10 and G6PD should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization.

中文翻译：

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通过定量实时 PCR 验证大豆基因表达研究的内部对照。


背景 对管家基因 (HKG) 进行标准化可以使定量实时 PCR (qRT-PCR) 的结果更加可靠。最近的研究表明，没有一个 HKG 可以适用于所有实验。因此，在使用之前应选择合适的HKG。只有少数关于植物中 HKG 的研究，并且没有针对大豆（一种重要的经济作物）的研究。因此，本研究旨在确定适合大豆基因表达标准化的 HKG。结果 所有 10 个 HKG 在 21 个样本池中都显示出广泛的 Ct 值，证实它们的表达存在差异。 GeNorm 用于确定七个系列组中 HGK 的表达稳定性。对于分析的所有样本池，稳定性排名为 ELF1B、CYP2 > ACT11 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10。对于同一发育阶段的不同组织，排序为ELF1B、CYP2>ACT2/7>UBC2>TUA>ELF1A>ACT11>TUB>G6PD>UBQ10。对于发育阶段系列，稳定性排名为ACT2/7，TUA > ELF1A > UBC2 > ELF1B > TUB > CYP2 > ACT11 > G6PD > UBQ10。对于光周期治疗，排名为ACT11、ELF1B > CYP2 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10。对于一天中的不同时间，排名为 ELF1A、TUA > ELF1B > G6PD > CYP2 > ACT11 > ACT2/7 > TUB > UBC2 > UBQ10。对于不同品种和主茎不同节上的叶子，10个HKG的稳定性没有显着差异。 Delta Ct 方法和“稳定性指数”也用于分析所有 21 个样本池的表达稳定性。 Delta Ct 方法和 geNorm 的结果表明，ELF1B 和 CYP2 是最稳定的 HKG，而 UBQ10 和 G6PD 是最可变的。 “稳定性指数”分析的结果不同，ACT11 和 CYP2 是最稳定的 HKG，ELF1A 和 TUA 是最不稳定的。结论 我们的数据表明 HKG 在大豆中的表达存在差异。根据geNorm和Delta Ct分析的结果，ELF1B和CYP2可以作为内参使大豆基因表达标准化，而应避免使用UBQ10和G6PD。为了获得准确的结果，某些情况可能需要使用多个 HKG 进行标准化。