Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction.
OBJECTIVE
There is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA.


METHODS
RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes.


RESULTS
In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization.


CONCLUSIONS
Numerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.
Neumann
E
E
University of Regensburg, Germany.
Kullmann
F
F
Judex
M
M
Jüsten
H P
HP
Wessinghage
D
D
Gay
S
S
Schölmerich
J
J
Müller-Ladner
U
U
D001172:Arthritis, Rheumatoid / Q000235:genetics*; D005347:Fibroblasts / Q000502:physiology; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D009857:Oncogenes / Q000235:genetics; D010003:Osteoarthritis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA; D013583:Synovial Membrane / Q000166:cytology
C034477:RNA primers; D012313:RNA