Laser‐mediated microdissection facilitates analysis of area‐specific gene expression in rheumatoid synovium

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Abstract
Objective Current approaches to analyzing gene expression in rheumatoid arthritis (RA) synovium are based on RNA isolated either from cultured synovial cells or from synovial biopsy specimens. This strategy does not, in general, allow distinction of specific gene expression between cells originating from different synovial areas, due to potential mixture of expression profiles. Therefore, we established the combination of laser‐mediated microdissection (LMM) and differential display to analyze profiles of gene expression in histologically defined areas of rheumatoid synovium. The present study was undertaken to establish parameters for this technique and assess its usefulness for gene expression analysis. Methods Cryosections derived from RA synovial tissues were used to obtain cell samples from synovial lining versus sublining, using a microbeam laser microscope. RNA was isolated and analyzed by nested RNA arbitrarily primed–polymerase chain reaction (RAP‐PCR) for differential display fingerprinting. Differentially expressed bands were cut out, and PCR products were eluted, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry analysis. Results Microdissected sections of RA synovial tissue containing ∼600 cells yielded enough RNA to produce a reproducible RNA fingerprint pattern. Several genes could be identified as being expressed differentially between the synovial lining and the sublining, and their expression could be confirmed at the messenger RNA and protein levels. Conclusion The combination of LMM and RAP‐PCR presents a valuable tool to obtain novel insights into the area‐dependent differential regulation of gene expression in RA synovium. Both known and previously unknown genes were revealed with this technique. This study is the first to demonstrate the potential of this analytic strategy in the investigation of a nonmalignant, multifactorial, inflammatory disease.