Research article

Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

Manuel Pombo-Suarez¹ , Manuel Calaza¹ , Juan J Gomez-Reino^1,2 and Antonio Gonzalez¹

¹Laboratorio de Investigacion 2 and Rheumatology Unit, Hospital Clinico Universitario de Santiago, Santiago de Compostela, Spain

²Department of Medicine, University of Santiago de Compostela, Spain

author email corresponding author email

BMC Molecular Biology 2008, 9:17doi:10.1186/1471-2199-9-17

Published:	29 January 2008

Abstract

Background

Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application.

Results

Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared.

Conclusion

Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.