title: Selection of suitable reference genes for normalization of quantitative real-time polymerase chain reaction in human cartilage endplate of the lumbar spine.
abstract: The quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression profiles, and it requires appropriate normalization for accurate and reliable results. Although several genes are commonly used as reference genes (such as GAPDH, ACTB, and 18S rRNA), they are also regulated and can be expressed at varying levels. In this study, we evaluated twelve well-known reference genes to identify the most suitable housekeeping gene for normalization of qRT-PCR in human lumbar vertebral endplate with Modic changes, by using the geNorm, NormFinder, and BestKeeper algorithms. Our results showed that the rarely-used SDHA was the most stable single reference gene, and a combination of three, SDHA, B2M, and LDHA, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, GAPDH, ACTB and 18S rRNA, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although SDHA and RPL13A uniformly ranked in the first and last position, respectively. Further simulated expression analysis validated that the arbitrary use of a reference gene could lead to the misinterpretation of data. Our study confirmed the necessity of exploring the expression stability of potential reference genes in each specific tissue and experimental situation before quantitative evaluation of gene expression by qRT-PCR.
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Zhou
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Zhang
authors.affiliation: Department of Neurology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Xia
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ji-Ying
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Xiang-Qian
authors.initials: XQ
authors.lastname: Fang
authors.affiliation: Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou Zhejiang, China ; Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Shun-Wu
authors.initials: SW
authors.lastname: Fan
mesh_terms: D002356:Cartilage / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D008159:Lumbar Vertebrae / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards