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 been 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 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. Differentially 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.
Zanders
E D
ED
Immunopathology Unit, Glaxo-Wellcome Research and Development, Gunnels Wood Road, Stevenage, UK. edz0107@glaxowellcome.com
Goulden
M G
MG
Kennedy
T C
TC
Kempsell
K E
KE
D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D016022:Case-Control Studies; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006801:Humans; D008264:Macrophages / Q000276:immunology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010950:Plasma Cells / Q000276:immunology; D013583:Synovial Membrane / Q000276:immunology
D017931:DNA Primers; D018076:DNA, Complementary