title: Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in Chilo suppressalis (Lepidoptera: Pyralidae).
abstract: Quantitative real-time polymerase chain reaction (qRT-PCR) is a valuable tool for estimating gene expression; however, the validity is largely dependent on the selection of stable reference genes. The suitability of various reference genes for qRT-PCR analysis was evaluated in, Chilo suppressalis (Walker). The ΔCt method, geNorm, NormFinder, and BestKeeper were used to evaluate the suitability of nine candidate reference genes for normalizing gene expression in larval tissues and organs and during high and low temperature stress. The ΔCt method, geNorm, and NormFinder produced similar stability rankings; H3, UBI, and EF1 were the most stable reference genes for monitoring gene expression in larval tissue and organs, and EF1, TUB, and AK were the optimal genes for thermal stress. However, for thermal stress, RPS11 was the most stable gene based on BestKeeper. To validate these recommendations, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. Hsp60 transcript levels showed significant differences when normalized to the most versus least stable reference genes. These results further confirm the importance of testing reference genes using the selected experimental parameters. The reference genes identified in the present study will improve the quality of gene expression data obtained for C. suppressalis and will facilitate future studies aimed at understanding the biology of this important insect pest.
authors.affiliation: School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou 225009, China ( tczxyt@gmail.com ; lumx@yzu.edu.cn ; yzdu@yzu.edu.cn ).|Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou 225009, China ( tczxyt@gmail.com ; lumx@yzu.edu.cn ; yzdu@yzu.edu.cn ).|Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China.
authors.forename: Ming-Xing
authors.initials: MX
authors.lastname: Lu
authors.affiliation: Department of Life Science, Fuyang Normal College, Fuyang 236032, China (fysyswx@163.com).
authors.forename: Ya-Dong
authors.initials: YD
authors.lastname: Cui
authors.affiliation: School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou 225009, China ( tczxyt@gmail.com ; lumx@yzu.edu.cn ; yzdu@yzu.edu.cn ).|Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China.
authors.forename: Yu-Zhou
authors.initials: YZ
authors.lastname: Du
mesh_terms: D000818:Animals; D003080:Cold Temperature; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D017344:Genes, Insect; D006358:Hot Temperature; D007814:Larva / Q000235:genetics / Q000254:growth & development; D009036:Moths / Q000235:genetics* / Q000254:growth & development; D009928:Organ Specificity; D060888:Real-Time Polymerase Chain Reaction; D013312:Stress, Physiological
keywords: Chilo suppressalis; evaluation; qRT-PCR; reference gene