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Identification of multiple, differentially expressed messenger RNAs in dermal fibroblasts from patients with systemic
1University of Pittsburgh, Pennsylvania 15261, USA.
Objective:
To simultaneously identify several genes whose expression is altered in dermal fibroblasts from patients with systemic sclerosis (SSc).
Methods:
Total RNA was prepared from fibroblasts derived from clinically affected and unaffected skin of patients with SSc. The RNA samples were analyzed using differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The differential expression of the corresponding mRNAs was confirmed by ribonuclease protection assay.
Results:
We identified 21 differentially expressed mRNAs. Their corresponding cDNAs were sequenced and the sequences obtained were compared with those of known genes entered into the EMBL/GenBank database. Three of the sequences corresponded to transcripts of yet-unidentified genes. Some of the mRNAs shared partial homology with extracellular matrix components, cellular receptors, enzymes, and nuclear factors. Others corresponded to known mRNAs such as those of fibronectin, fibronectin receptor, laminin receptor homolog, beta-tubulin, insulin-like growth factor binding protein 5, KIAA0179 protein, and protease nexin 1.
Conclusion:
The application of DDRT-PCR to scleroderma research has identified many mRNAs whose altered expression in scleroderma has not yet been described, thus providing new information for further investigation and potential targets for the development of novel therapies.