EXTENDED REPORT

Technical validation of cDNA based microarray as screening technique to identify candidate genes in synovial tissue biopsy specimens from patients with spondyloarthropathy

M Rihl^1,3, D Baeten², N Seta³, J Gu³, F De Keyser², E M Veys², J G Kuipers¹, H Zeidler¹, D T Y Yu³

¹ Hannover Medical School, Department of Rheumatology, Hannover, Germany
² Ghent University Hospital, Department of Rheumatology, Ghent, Belgium
³ University of California Los Angeles, UCLA School of Medicine, Division of Rheumatology, Los Angeles, CA, USA

Correspondence to:
Correspondence to:
Dr M Rihl
Hannover Medical School (MHH), Department of Rheumatology (OE 6850), Carl-Neuberg-Str. 1, 30625 Hannover, Germany; Rihl.Markus{at}MH-Hannover.de

Objectives: To validate the use of cDNA based microarray on synovial biopsies by analysing the experimental variability due to amplification of RNA, reproducibility of the assay, heterogeneity of the tissue, and statistical analysis.

Methods: Total RNA was extracted from three spondyloarthropathy (SpA) and three osteoarthritis (OA) synovial tissue biopsy specimens and from the peripheral blood mononuclear cells (PBMC) of four healthy donors. Exponential RNA amplification by SMART-PCR was compared with linear amplification. Reproducibility was tested by comparing different microarray systems and by performing duplicate experiments. Sample heterogeneity was assessed by comparing overall gene expression profiles, histopathology, and analysis of genes expressed in the synovium and normal PBMC. Statistical analysis using t test and Bonferroni adjustment was verified by permutation of class labels.

Results: Gene expression was concordant in 12/14 (86%) cytokine/chemokine genes between both microarrays and different RNA amplification systems. When one microarray system was used, expressed genes were 78–95% concordant in duplicate experiments. Gene expression profiles had a higher degree of similarity between SpA synovium than between PBMC or OA synovium despite clear histopathological differences between synovial samples. Comparison of SpA synovium with OA synovium and with PBMC yielded 11 and 18 expressed transcripts, respectively; six were shared in both comparisons. Permutations of SpA and OA samples yielded only one expressed gene in 19 comparisons.

Conclusions: These data provide evidence that microarrays can be used for analysis of synovial tissue biopsies with high reproducibility and low variability of the generated gene expression profiles.

Keywords: microarray; SMART-PCR; spondyloarthropathies; synovial tissue; peripheral blood mononuclear cells

Abbreviations: DMARDs, disease modifying antirheumatic drugs; HC, healthy controls; LPS, lipopolysaccharide; OA, osteoarthritis; PBMC, peripheral blood mononuclear cells; RT-PCR, reverse transcriptase-polymerase chain reaction; SMART, Switch Mechanism At the 5' end of the RNA Template; SpA, spondyloarthropathy

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Wilhelm, J., Muyal, J. P., Best, J., Kwapiszewska, G., Stein, M. M., Seeger, W., Bohle, R. M., Fink, L. (2006). Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments. Clin. Chem. 52: 1161-1167 [Abstract] [Full Text]  
• Smith, M D, Baeten, D, Ulfgren, A-K, McInnes, I B, Fitzgerald, O, Bresnihan, B, Tak, P P, Veale, D, on behalf of the OMERACT synovial special interest, (2006). Standardisation of synovial tissue infiltrate analysis: how far have we come? how much further do we need to go?. Ann Rheum Dis 65: 93-100 [Abstract] [Full Text]  
• Rihl, M, Kruithof, E, Barthel, C, De Keyser, F, Veys, E M, Zeidler, H, Yu, D T Y, Kuipers, J G, Baeten, D (2005). Involvement of neurotrophins and their receptors in spondyloarthritis synovitis: relation to inflammation and response to treatment. Ann Rheum Dis 64: 1542-1549 [Abstract] [Full Text]  
• Rihl, M, Gu, J, Baeten, D, Marker-Hermann, E, Goodall, J C, Gaston, J S H, Kuipers, J G, Zeidler, H, Yu, D T Y (2004). Alpha beta but not gamma delta T cell clones in synovial fluids of patients with reactive arthritis show active transcription of tumour necrosis factor {alpha} and interferon {gamma}. Ann Rheum Dis 63: 1673-1676 [Abstract] [Full Text]