Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays

Subtractive hybridization of cDNAs generated from synovial RNA which had be en isolated from patients with 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 exp ression 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 drive r cDNA, and subjected to suppression subtraction PCR. Differentially expres sed products were cloned into E. coli and picked into 384 well plates. Inse rts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also l abelled by random priming for use as probes for library screening. The libr aries chosen were the subtracted one described above and a set of 45,000 ES Ts from the I.M.A.G.E consortium. Clones showing positive hybridization wer e identified by sequence analysis and homology searching. The results showe d that the subtracted hybridization approach could identify many gene fragm ents expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cel l and plasma cell infiltration with evidence of interferon induction. In ad dition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in fi nding novel gene fragments for further characterisation as potential member s of the immune system. Although RA was studied here, the technology is app licable to any disease process even in cases where amounts of tissue may be limited. (C) 2000 Elsevier Science B.V. All rights reserved.