Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR.
Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR.
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DOI：
10.1186/1471-2199-10-93
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发表时间：
2009-09-28
影响因子：
--
通讯作者：
Fu YF
中科院分区：
文献类型：
--
作者：
Hu R;Fan C;Li H;Zhang Q;Fu YF
实时定量逆转录PCR（RT-qPCR）数据需要进行标准化，以便正确解释。管家基因通常用于此目的，但它们的表达水平不能假定在所有可能的实验条件下保持恒定。因此，需要对参考基因进行系统验证以确保适当的标准化。对于大豆，迄今为止只有少数经过验证的参考基因可用。对14个大豆潜在参考基因进行了系统比较。其中包括7个常用的（ACT 2、ACT 11、TUB4、TUA5、UBQ、UBQ10、EF1b）和7个新的候选者（SKIP 16、MTP、PEPKR1、HDC、TIP41、UKN1、UKN2）。通过RT-qPCR在116个生物样品中检查表达稳定性，这些生物样品代表不同发育阶段的组织、不同的光周期处理和一系列大豆栽培品种。所有14个基因的表达在一定程度上是可变的，但SKIP 16、UKN1和UKN2的表达总体上是最稳定的。ACT11、UKN1和UKN2的组合将适合作为标准化不同组织中基因表达数据的参考组，而SKIP16、UKN1和MTP的组合最适合于发育阶段。ACT 11、TUA5和TIP41在光周期改变时表达最稳定，TIP41、UKN1和UKN2在光质改变时表达最稳定。6个不同品种在长日照和短日照条件下的表达稳定性差异不显著，其中ACT 11、UKN2和TUB4基因的表达稳定性最高。本研究以拟南芥FT基因的同源基因GmFTL 3（FLOWESTERN LOCUS T）为参考基因，通过基因表达水平的测定，验证了所选参考基因的正确性。没有一个候选参考基因在所有实验条件下均匀表达，并且最合适的参考基因是条件依赖性的、组织特异性的、发育依赖性的和栽培依赖性的。大多数新的参考基因比传统的管家基因表现得更好。这些结果将指导大豆基因表达研究中参考基因的选择。
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. A 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. 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.
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影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
9.2
作者：
Jaeger, Katja E.;Wigge, Philip A.
通讯作者：
Wigge, Philip A.
影响因子：
--
作者：
Jung M;Ramankulov A;Roigas J;Johannsen M;Ringsdorf M;Kristiansen G;Jung K
通讯作者：
Jung K
影响因子：
--
作者：
Ahn K;Huh JW;Park SJ;Kim DS;Ha HS;Kim YJ;Lee JR;Chang KT;Kim HS
通讯作者：
Kim HS
影响因子：
3.5
作者：
Bustin, SA
通讯作者：
Bustin, SA