Int J Biol Sci 2013; 9(8):792-802. doi:10.7150/ijbs.5862
Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)
1. Institute of Pesticide Science, Hunan Agricultural University, Changsha 410128, China.
2. Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
3. Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China.
4. Key Laboratory of Integrated Management of the Pests and Disease on Horticultural Crops in Hunan Province, Changsha 410125, China.
5. Department of Entomology, University of Kentucky, Lexington, KY 40546-0091, U.S.A.
Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest.
Keywords: Plutella xylostella, reference gene, qRT-PCR analysis, biotic factor, abiotic factor.
Fu W, Xie W, Zhang Z, Wang S, Wu Q, Liu Y, Zhou X, Zhou X, Zhang Y. Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae). Int J Biol Sci 2013; 9(8):792-802. doi:10.7150/ijbs.5862. Available from http://www.ijbs.com/v09p0792.htm