Objective: To screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS).
Methods: We constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed.
Results: The forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively.
Conclusions: Two positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.