Identifying Novel Genes In Endometriosis Using Representational Difference Analysis Of CDNA

Identifying novel genes in endometriosis using representational difference analysis of cDNAObjectiveEMs(endometriosis) is an estrogen dependent disease commonly among the women in reproductive age, and has a significantly increasing tendency of morbility. Recent researches indicate that EMs is relative to genetic factors and disorders of genes. But the real cause of it is still unclear. Some believe that there is phenotypic difference between heredity and gene. Some believe that endometrium is composed of kinds of heterogenetic cellular subset with different reactiveness. The one having strong proliferating capacity of the retrograding menstrual endometrium may develop to EMs, and it may be also the result of disturbance in growth regulation according to environment changes. In recent years, many researchers find that genes and protein expression are different between ectopic endometrium and normal one. Our study is focused on EMs-related genes. We adopt cDNA representational difference analysis to confirm the genes which may contribute to expose the pathogenesis of diseases in gene level and explore the effective methods to cure the diseases.Methods1,cDNA-RDA,Cloning and SequencingTotal RNAs were extrated from the samples mentioned above Trizol reagent, mRNA were esolated by mRNA purification kit,Double strands cDNA were synthesized then cut by Sau3A1, ligated to R-12/24 adaptor after purification. Oligonucleotides were annealed to each other,then incubated for 12h at 12-16℃. Multiple PCR reactions were set up to generate the initial representations after dilution, The R-adaptors were removed from the representations with the same endonuclease, and the digest was phenol extracted and ethol precipitated to form driver, and the tester was ligated to the J-12/24 adaptor in the manner described above. The first subtractive hybridization was carried out at the ratio of 1:100 in 4 ul EPPS buffer at 67℃for 20h, then the diluted hybridization mixure was amplified, 18cycles, the 24bp adaptors acted as primers, then got the first differential products. The latter two rounds of subtractive hybridization were performed at the ratio of 1:400 and 1:80000 respectively, DPⅢwas composed of many fragments which were purified and cloned into pMD18-vector. The clonings were then sequenced by Lian-he gene company in Shanghai after confirmed by PCR.2,RT-PCR TestEutopic endometrium was obtained from 10 non-endometriosisPatients, 7 endometriums of endometriosis are to be paired. It probed that the different fragments originated from mRNA.RESULT1,cDNA-RDA, identifying and sequencingWe obtained one different cDNA fragments, which then were purified and cloned into pMD-18-vestor. The recombinated vector was transformed into Top 10 competent cells. The passitive clones were sequenced, then we analysis the similarity of Nucleic Acid and Amino Acid with Blast similarity searching system in MCBI. Squence comparision revealved 99% respectively homology of DPⅢwith cofilin.2,RT-PCR TestThe different fragments originated from mRNA. And it is up-graduat obviously endometriosis tissue.ConclusionFirstly studied the novel genes related to endometriosis using cDNA-Representational Difference Analysis and screened one genes fragments, which had homology with cofilin. It was shown that cofilin has a higher regulation in diagnosis endometriosis due to changes brought about genetic and immunologic factors(p＜0.05). Can this be brought about the secondary changes in the histology or the primary changes? In this experiment, we find 11 different gene sequences, 7 can't be found out the same source,probably they are novel genes. At the same time, research on the biological molecules of endometriosis provide us a new phenomena with the structure and function.