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 transferred with probes of reverse transcription products cDNA of gastric cancer tissues mRNA and counterpart normal gastric mucosa mRNA labeled with alpha- (32)P 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.