Objective: To compare gene express difference of keloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so as to find the differential express gene in keloid.
Methods: mRNA extracted from keloid and normal skin tissues was used as the template to synthesis cDNA of keloid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with Rsa I. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplification were done. Differentially expressed cDNA was selectively amplified during these reactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones of Southern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank.
Results: Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function.
Conclusion: Keloid differentially expressed gene was screened successfully by SSH.