Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

Yong Li, You-Yong Lu, Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, Beijing 100034, China

Author contributions: All authors contributed equally to the work.

Correspondence to: Dr. You-Yong Lu, Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, 1 Da-Hong-Luo-Chang Street, Western District, Beijing 100034, nc.ten.atb.cilbup@ulygnoy.anihC

Telephone: +86-10-66163061 Fax: +86-10-66175832

Yong Li, You-Yong Lu, Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, Beijing 100034, China

Author contributions: All authors contributed equally to the work.

Telephone: +86-10-66163061 Fax: +86-10-66175832

Received 2001 Sep 14; Revised 2001 Oct 15; Accepted 2001 Oct 21.

Abstract

AIM: To develop and optimize cDNA representational difference 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 two self-constructed cDNA libraries (Allitridi-treated and paternal HGC 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 and Slot blots analyses were employed to identify the obtained difference products.

RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-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 cells after 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.

Abstract

Footnotes

Edited by Hu DK

Supported by the Natural Scientific Foundation of China (NSFC3962526) and National High-Technology Project-863 (102-10-01-04)

Footnotes

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