Objective: To identify genes that are differentially expressed in systemic sclerosis (SSc) fibroblasts of clinically involved and noninvolved skin compared to normal dermal fibroblasts, using RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) for differential display.
Methods: We examined 12 fibroblast cultures derived from clinically involved skin, 3 fibroblast cultures from noninvolved skin, and 4 fibroblast cultures from healthy skin. After extraction of total RNA, the first step of RAP-PCR was performed using different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling using different arbitrary 10-base primers, followed by sequence analysis of the amplified fingerprint products. The resulting sequences were aligned to the GenBank database using Blast Search. Confirmation of differential expression was performed with specific primers using real-time PCR.
Results: Using 8 different primer combinations, in total 48 cDNA were differentially expressed between SSc and healthy dermal fibroblasts. Sequence analysis identified distinct PCR products, which were overexpressed in SSc as highly homologous to gene segments of gremlin protein, lysyl oxidase, c-cbl proto-oncogene, an estrogen-responsive element, fibronectin, and collagen type XIIa1 precursor.
Conclusion: Our results show that RAP-PCR is a suitable method to identify differentially expressed genes in SSc fibroblasts. Further, we identified genes that have not yet been described in the pathophysiology of SSc and that may be involved in matrix synthesis and cellular interaction.