title: Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress.
abstract: Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H2O2) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H2O2-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H2O2treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.
authors.affiliation: a Department of Ophthalmology , The Second Hospital of Jilin University, Jilin University , Changchun , PR China.|b Department of Ophthalmology , China-Japan Union Hospital, Jilin University , Changchun , PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: b Department of Ophthalmology , China-Japan Union Hospital, Jilin University , Changchun , PR China.
authors.forename: HongYan
authors.initials: H
authors.lastname: Zhou
authors.affiliation: a Department of Ophthalmology , The Second Hospital of Jilin University, Jilin University , Changchun , PR China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Sun
authors.affiliation: b Department of Ophthalmology , China-Japan Union Hospital, Jilin University , Changchun , PR China.
authors.forename: Ben
authors.initials: B
authors.lastname: Yang
authors.affiliation: b Department of Ophthalmology , China-Japan Union Hospital, Jilin University , Changchun , PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: c Department of Rehabilitation , China-Japan Union Hospital, Jilin University , Changchun , PR China.
authors.forename: Hongfeng
authors.initials: H
authors.lastname: Shi
authors.affiliation: a Department of Ophthalmology , The Second Hospital of Jilin University, Jilin University , Changchun , PR China.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Zheng
mesh_terms: D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000592:standards*; D020043:Genes, Essential; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D014129:Trabecular Meshwork / Q000166:cytology
chemical_list: D016877:Oxidants; D006861:Hydrogen Peroxide
keywords: H2O2; Oxidative stress; glaucoma; reference gene; trabecular meshwork cell