title: Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae).
abstract: To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.
authors.affiliation: Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Changsha, Hunan, China; Department of Entomology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Entomology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Huipeng
authors.initials: H
authors.lastname: Pan
authors.affiliation: Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Changsha, Hunan, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Entomology, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Zhou
mesh_terms: D000818:Animals; D001042:Aphids / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D017344:Genes, Insect; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012016:Reference Values; D012984:Software