Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.
Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.
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DOI：
10.1673/031.012.6001
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Li F
中科院分区：
文献类型：
--
作者：
Teng X;Zhang Z;He G;Yang L;Li F
实时定量聚合酶链式反应 (qPCR) 是一种有效且广泛使用的基因表达监测技术。管家基因（HKG）通常根据经验选择作为数据标准化的参考基因。然而，HKGs 用作内参基因的适用性很少得到验证。在此，在四种鳞翅目昆虫 Bombyx mori L.（鳞翅目：Bombycidae）、Plutella xylostella L.（Plutellidae）、Chilo Preventalis Walker（Crambidae）和 Spodoptera exigua Hübner 中选择了 6 个 HKG（肌动蛋白 A3、肌动蛋白 A1、GAPDH、G3PDH、E2F、rp49）。 （Noctuidae）研究其表达稳定性。使用geNorm、NormFinder、稳定性指数和ΔCt分析算法来评估这些HKG。在不同发育阶段，肌动蛋白A1在小菜蛾和二化螟中最稳定，而在家蚕和甜菜夜蛾中最不稳定。 Rp49 和 GAPDH 分别在桑蚕和甜菜夜蛾中最稳定。在不同组织中，GAPDH、E2F和Rp49分别在家蚕、甜菜夜蛾和二化螟中最稳定。以不同的HKG为内参基因测试了2-ΔΔCt法估算的Siwi基因的相对丰度，证明了内参在qPCR数据分析中的重要性。结果不仅提供了四种鳞翅目昆虫中合适的参考基因列表，而且证明了HKGs的表达稳定性在进化上接近的物种之间存在差异。没有一个通用的参考基因可以在所有情况下使用。在使用 HKG 作为 qPCR 的内部对照之前，验证 HKG 的表达是必不可少的。
Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2-ΔΔCt method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.
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影响因子：
--
作者：
Silver N;Best S;Jiang J;Thein SL
通讯作者：
Thein SL
影响因子：
4
作者：
Wang, Gen-Hong;Xia, Qing-You;Zhu, Li
通讯作者：
Zhu, Li
影响因子：
3.3
作者：
Selvey, S;Thompson, EW;Griffiths, LR
通讯作者：
Griffiths, LR
影响因子：
13.8
作者：
Gutierrez, Laurent;Mauriat, Melanie;Van Wuytswinkel, Olivier
通讯作者：
Van Wuytswinkel, Olivier
影响因子：
56.9
作者：
Walker, NJ
通讯作者：
Walker, NJ