Ž . Journal of Immunological Methods 233 2000 131–140 www.elsevier.nlrlocaterjim Recombinant Technology Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays Edward D. Zanders a, ) , Matthew G. Goulden a , Tracy C. Kennedy b , Karen E. Kempsell a a Immunopathology Unit, Glaxo-Wellcome Research and DeÕelopment, Gunnels Wood Road, SteÕenage, Herts SG1 2NY, UK b Genetics Directorate, Glaxo-Wellcome Research and DeÕelopment, Gunnels Wood Road, SteÕenage, Herts SG1 2NY, UK Received 27 April 1999; received in revised form 8 July 1999; accepted 16 August 1999 Abstract Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with Ž . rheumatoid arthritis RA or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differen- tially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M.A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Subtractive hybridization; Rheumatoid arthritis; Expressed genes AbbreÕiations: RA, rheumatoid arthritis; Nor, normal; PCR, polymerase chain reaction; EST, expressed sequence tag; I.M.A.G.E., molecular analysis of genomes and their expression ) Corresponding author. Tel.: q44-1438-745745; fax: q44-1438-764263; e-mail: edz0107@glaxowellcome.com 0022-1759r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 99 00126-X