Selection of suitable reference genes for normalization of quantitative real-time polymerase chain reaction in human cartilage endplate of the lumbar spine

Zhi-Jie Zhou; Jian-Feng Zhang; Ping Xia; Ji-Ying Wang; Shuai Chen; Xiang-Qian Fang; Shun-Wu Fan

doi:10.1371/journal.pone.0088892

Selection of suitable reference genes for normalization of quantitative real-time polymerase chain reaction in human cartilage endplate of the lumbar spine

PLoS One. 2014 Feb 18;9(2):e88892. doi: 10.1371/journal.pone.0088892. eCollection 2014.

Authors

Zhi-Jie Zhou¹, Jian-Feng Zhang¹, Ping Xia², Ji-Ying Wang¹, Shuai Chen¹, Xiang-Qian Fang¹, Shun-Wu Fan¹

Affiliations

¹ 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.
² 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cartilage / metabolism*
Gene Expression Profiling
Humans
Lumbar Vertebrae / metabolism*
Real-Time Polymerase Chain Reaction / standards*
Reference Standards

Grants and funding

The study was supported by the National Natural Science Fund of China (81301587, 81271971, 81171739, 81101378), the Natural Science Fund of Zhejiang Province (Y2110372, LQ13H060002), and the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.