Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints

Abstract

Objective: To quantify the expression of genes encoding extracellular matrix (ECM) proteins in human cartilage from normal and osteoarthritic (OA) joints.

Design: Human cartilage samples were classified as control (CTR) or OA according to clinical evaluation and assessed histologically and biochemically to confirm the diagnosis. mRNAs encoding collagen types I, II and X, aggrecan, versican, osteopontin and osteocalcin were quantified by real-time reverse transcription-PCR assays and normalized to a reference mRNA (GAPDH).

Results: RNA from native cartilage could be reproducibly and efficiently amplified by real-time PCR only if isolated using purification membranes. Primers and fluorescent probes for real-time PCR, endowed with comparable (<6% difference from GAPDH) and high (>91%) amplification efficiencies, were designed and validated for the selected ECM genes. The expression of most genes under investigation displayed large variations and was not significantly different in CTR and OA cartilage. Only osteopontin mRNA levels were significantly higher in OA than CTR specimens. mRNA ratios of collagen type II to I and of aggrecan to versican, defined as indexes of chondrocyte differentiation, were less variable within each population than the single genes and markedly higher (27.0 and 7.6-fold, respectively) in CTR than OA cartilage, with high statistical significance (P = 0.00013 and P = 0.00007, respectively).

Conclusions: Our results provide evidence that gene patterns related to chondrocyte differentiation discriminate between CTR and OA human cartilage with higher sensitivity than single ECM genes. The method described here has the potential to improve understanding of the progression of OA and could become a valuable diagnostic tool.