Genotyping of synovial fibroblasts: cDNA array in combination with RAP-PCR in arthritis.
Evaluation of differentially regulated genes is essential for the development of novel therapeutic approaches in multifactorial diseases such as rheumatoid arthritis (RA). RA synovial fibroblasts (RASF) are key players in inflammation and cartilage destruction. Therefore, RASF are important cellular targets for the analysis of gene expression profiles. Such analyses may include a comparison of SF from nontreated RA patients with those from treated RA patients, and may also be used to evaluate which genes and intra-cellular processes can be modulated through genetic modification, gene transfer, and drug treatment for the identification of the pathways driving the destructive behavior of these cells. This chapter reports the combination of RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and cDNA array with defined genes for a highly sensitive analysis of gene expression profiles in RASF using small amounts of total RNA. RNA can be extracted from cultured SF, isolated, and analyzed using RAP-PCR with different arbitrary primers for first-and second-strand synthesis to generate a radioactive labeled probe which can be used for cDNA array hybridization. Visualization and evaluation of gene expression can be performed by phosphorimaging in combination with an array-specific software analyzing system followed by statistic evaluation of the generated data. In summary, the combination of RAP-PCR combined with cDNA arrays is a sensitive method to identify differentially expressed genes in RASF with high specificity, especially for low abundant mRNAs.
Neumann
Elena
E
Judex
Martin
M
Gay
Steffen
S
Müller-Ladner
Ulf
U
D001168:Arthritis / Q000235:genetics* / Q000473:pathology; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data; D010003:Osteoarthritis / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology
D018076:DNA, Complementary; D012313:RNA