Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
J Insect Sci. 2012;12:60. doi: 10.1673/031.012.6001.
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.
实时荧光定量聚合酶链反应（qPCR）是一种高效且广泛使用的技术，用于监测基因表达。管家基因（HKG）通常被经验性地选为数据归一化的参考基因。然而，用作参考基因的 HKG 的适用性很少得到验证。在这里，选择了六个 HKG（肌动蛋白 A3、肌动蛋白 A1、GAPDH、G3PDH、E2F、rp49）在四个鳞翅目昆虫家蚕（鳞翅目：蚕科）、小菜蛾（菜蛾科）、斜纹夜蛾（斜纹夜蛾科）和甜菜夜蛾（夜蛾科）中研究其表达稳定性。使用 geNorm、NormFinder、稳定性指数和ΔCt 分析算法来评估这些 HKG。在不同的发育阶段，肌动蛋白 A1 在小菜蛾和斜纹夜蛾中最稳定，但在家蚕和甜菜夜蛾中最不稳定。Rp49 和 GAPDH 在 B. mori 和 S. exigua 中最稳定。在不同的组织中，GAPDH、E2F 和 Rp49 在 B. mori、S. exigua 和 C. suppressalis 中最稳定。通过 2(-ΔΔCt)方法估计的 Siwi 基因的相对丰度用不同的 HKG 作为参考基因进行了测试，证明了内部对照在 qPCR 数据分析中的重要性。结果不仅提供了四个鳞翅目昆虫中合适的参考基因列表，还证明了 HKG 的表达稳定性在进化上相近的物种之间存在差异。没有一个单一的通用参考基因可以在所有情况下使用。在 qPCR 中使用 HKG 作为内参之前，验证 HKG 的表达是必不可少的。
