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AIM:To develop and optimize cDNA representationaldifference analysis(cDNA RDA)method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC)cells.METHODS:We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries(Allitridi-treated and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH I and Xho I restriction sites harbored in the libraryvector were used to select representations,Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESULTS:Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA.Two novel expressed sequence tags(ESTs)and arecombinant gene were obtained.Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1(GDN/PN1)gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein(XIP)mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic’s efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
AIM: To develop and optimize cDNA representational discrimination analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by twoself- constructed cDNA libraries (Allitridi-treated and paternalHGC cell line BGC823 cells cDNA libraries respectively) .BamH I and Xho I restriction sites harbored in the library vector were used to select representations, Northern andSlot blots analyzes were employed to identify the obtained resolution products .RESULTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as good marker prior the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant genes were obtained. Slot blots result showed a 8-fold increase of gila-derived nexin / protease nexin 1 (GDN / PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h. CONCLUSION: Elevated levels of GDN / PN1 and XIP mRNAs induced by Allitridi offered valuable molecular evidence for elucidating the garlic’s efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.