Gastric Cancer
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1439-1443
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1439
cDNA suppression subtraction library for screening down-regulated genes in gastric carcinoma
Jian-Jun Du, Ke-Feng Dou, Shu-You Peng, Hua-Sheng Xiao, Wei-Zhong Wang, Wen-Xian Guan, Zhong-Hua Wang, Zhi-Qing Gao, Ying-Bin Liu
Jian-Jun Du, Ke-Feng Dou, Wei-Zhong Wang, Wen-Xian Guan, Zhong-Hua Wang, Zhi-Qing Gao, Department of General Surgery, Xijing Hospital, The Fourth Military Medical University, Xian 710032, Shaanxi Province, China
Hua-Sheng Xiao, Department of Neurobiology, The Fourth Military Medical University, Xi an 710032, Shaanxi Province, China
Shu-You Peng, Ying-Bin Liu, Department of General Surgery, the Second Affiliated Hospital, Medical College, Zhejiang University, Hangzhou 310009, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Ke-Feng Dou, Department of General Surgery, Xijing Hospital, The Fourth Military Medical University, Xian 710032, Shaanxi Province, China. zhongmrh@yahoo.com
Telephone: +86-029-3375256
Received: January 4, 2003
Revised: February 4, 2003
Accepted: February 16, 2003
Published online: July 15, 2003
Abstract

AIM: To establish cDNA suppression subtraction library with a high subtraction efficiency by counterpart normal gastric mucosa mixture mRNA subtracting gastric cancer cells mixture mRNA for screening down-regulated genes in gastric carcinoma.

METHODS: RNA of gastric cancer tissues and counterpart normal gastric mucosa were respectively isolated in five patients with gastric cancer, and their mRNA was purified. cDNA suppression subtraction library was established by counterpart normal gastric mucosa mixture mRNA (tester) subtracting gastric cancer tissues mixture mRNA (driver) of five patients with gastric carcinoma. The library plasmids were transformed into competent bacteria DH5a after ligation of the library cDNA fragments with T vectors. Library plasmids were extracted after picking colonies and shaking bacteria overnight. Its subtraction efficiency was confirmed by PCR and reverse hybridization of a nylon filter onto which the colonies of bacteria were transfered with probes of reverse transcription products cDNA of gastric cancer tissues mRNA and counterpart normal gastric mucosa mRNA labeled with α-32P dCTP.

RESULTS: mRNA purified from total RNA of gastric cancer tissues and counterpart normal gastric mucosa in five patients with gastric carcinoma revealed a good quality. cDNA suppression subtraction library constructed for screening down-regulated genes in gastric carcinoma represented a high subtraction efficiency. 86% of differential expression in down-regulated genes between counterpart normal gastric mucosa and gastric carcinoma was confirmed.

CONCLUSION: cDNA suppression subtraction library with a high subtraction efficiency for screening down-regulated genes in gastric carcinoma is successfully established.

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