Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

• Published in: Journal of insect science (Tucson, Ariz.) Vol. 12; no. 60; pp. 1 - 17
• Main Authors: Teng, Xiaolu, Zhang, Zan, He, Guiling, Yang, Liwen, Li, Fei
• Format: Journal Article
• Language: English
• Published: United States University of Wisconsin Library 2012; Oxford University Press
• Subjects: actin; Actins - genetics; Actins - metabolism; algorithms; Animals; Bombycidae; Bombyx mori; Chilo suppressalis; Crambidae; developmental stages; E2F4 Transcription Factor - genetics; E2F4 Transcription Factor - metabolism; essential genes; expression stability; Gene expression; Genes, Essential; Genes, Insect; Genetic aspects; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Glycerolphosphate Dehydrogenase - genetics; Glycerolphosphate Dehydrogenase - metabolism; housekeeping gene; insects; Lepidoptera; Life Cycle Stages; Moths - genetics; Moths - growth & development; Moths - metabolism; Noctuidae; Plutella xylostella; Plutellidae; Polymerase chain reaction; qPCR; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction; reference gene; reverse transcriptase polymerase chain reaction; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; Spodoptera exigua; tissues; Varieties; United States
• ISSN: 1536-2442; 1536-2442
• 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.