title: Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori.
abstract: In general, for real-time quantitative polymerase chain reaction (qPCR), normalization strategies use a reference gene as a control and to avoid the introduction of experimental errors expression of this gene should not vary in response to changing conditions. However, the expression of many reference genes has been reported to vary considerably and, without appropriate normalization, the expression profile of a target gene can be misinterpreted. In this study, the expression levels of seven commonly used reference genes (ACT, GAPDH, 28srRNA, RPL3, α-tubulin, UBC, and TBP) were detected at different development time points and in response to treatment with 20-hydroxyecdysone (20E) and with rutin. The expression stability was analyzed using geNorm and NormFinder software. Significant variations were found among normal tissues and between experimentally treated tissues. The dual spike-in strategy also revealed significant variations of the expression levels of the reference genes among normal tissues and between experimentally treated tissues. Glutathione-S-transferase sigma 1 (GSTs1), which has a high expression level in fat body and is related to the mechanism of resistance, was used as a target gene to validate the feasibility and difference of these two approaches.
authors.affiliation: School of Biology and Basic Medical Sciences, Soochow University, Suzhou, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Peng
authors.forename: Yuanfen
authors.initials: Y
authors.lastname: Zhai
authors.forename: Hua
authors.initials: H
authors.lastname: Ding
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Di
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Weide
authors.initials: W
authors.lastname: Shen
authors.forename: Zhengguo
authors.initials: Z
authors.lastname: Wei
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000254:growth & development; D004441:Ecdysterone / Q000494:pharmacology; D005216:Fat Body / Q000378:metabolism; D020869:Gene Expression Profiling / Q000592:standards*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005982:Glutathione Transferase / Q000235:genetics; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics*; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics; D012431:Rutin / Q000494:pharmacology; D035181:TATA-Box Binding Protein / Q000235:genetics; D014404:Tubulin / Q000235:genetics; D025822:Ubiquitin C / Q000235:genetics
chemical_list: D000199:Actins; D019476:Insect Proteins; D012339:RNA, Ribosomal, 28S; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D035181:TATA-Box Binding Protein; D014404:Tubulin; D025822:Ubiquitin C; D004441:Ecdysterone; D012431:Rutin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D005982:Glutathione Transferase