title: Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae).
abstract: BACKGROUND
  Recent studies have focused on determining functional genes and microRNAs in the pest Helicoverpa armigera (Lepidoptera: Noctuidae). Most of these studies used quantitative real-time PCR (qRT-PCR). Suitable reference genes are necessary to normalize gene expression data of qRT-PCR. However, a comprehensive study on the reference genes in H. armigera remains lacking.
  
  
  RESULTS
  Twelve candidate reference genes of H. armigera were selected and evaluated for their expression stability under different biotic and abiotic conditions. The comprehensive stability ranking of candidate reference genes was recommended by RefFinder and the optimal number of reference genes was calculated by geNorm. Two target genes, thioredoxin (TRX) and Cu/Zn superoxide dismutase (SOD), were used to validate the selection of reference genes. Results showed that the most suitable candidate combinations of reference genes were as follows: 28S and RPS15 for developmental stages; RPS15 and RPL13 for larvae tissues; EF and RPL27 for adult tissues; GAPDH, RPL27, and β-TUB for nuclear polyhedrosis virus infection; RPS15 and RPL32 for insecticide treatment; RPS15 and RPL27 for temperature treatment; and RPL32, RPS15, and RPL27 for all samples.
  
  
  CONCLUSIONS
  This study not only establishes an accurate method for normalizing qRT-PCR data in H. armigera but also serve as a reference for further study on gene transcription in H. armigera and other insects.
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Songdou
authors.initials: S
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Wheat and Maize Crop Science (College of Plant Protection), Henan Agricultural University, Zhengzhou 450002, China.
authors.forename: Shiheng
authors.initials: S
authors.lastname: An
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Fengming
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Qingpo
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Yichen
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Huaijiang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China.
authors.forename: Qingwen
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Entomology, China Agricultural University, Beijing 100193, China. Electronic address: liuxiaoxia611@cau.edu.cn.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007306:Insecticides / Q000737:chemistry; D007814:Larva / Q000378:metabolism; D007915:Lepidoptera / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics; D013696:Temperature; D013879:Thioredoxins / Q000235:genetics
chemical_list: D007306:Insecticides; D035683:MicroRNAs; D012335:RNA, Ribosomal; D013879:Thioredoxins; D013482:Superoxide Dismutase
keywords: Abiotic conditions; Biotic conditions; Helicoverpa armigera; Normalization; Reference gene; qRT-PCR analysis