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Molecular Cloning of Human Stomach Carcinoma Metastasis Related Genes by Silver Stained RNA AP PCR Differential Display

Objective: To identify and clone differentially expressed genes in human primary and metastatic stomach carcinomas with improved silver stained RNA arbitrarily primed PCR method (RNA AP PCR). Methods: The total RNA populations which were respectively extracted from primary or metastatic stomach carcinoma tissue were firstly reverse transcribed with "anchor primer" olig(dT)12MN. Synthesized cDNA pools were then subjected to AP PCR amplification with 10 nt arbitrarily primers. Amplified products were resolved by electrophoresis on non denatural polyacrylamide gel and the differentially expressed PCR fragments were separated from silver stained gel. After RNA hybridization, T A vector cloning and DNA sequencing, the DNA sequences of fragments were checked with GenBank data for homology. Results: With improved silver stained RNA AP PCR, several differentially expressed PCR fragments were separated from primary or metastatic stomach carcinoma sample. Two fragments MGA1(662 bp) and PGA1 (589 bp) were reamplified and finally sequenced. The results showed that MGA1 was highly expressed in metastatic stomach carcinoma tissues and is complete homology to oncogene c fms, a gene coding human macrophage colony stimulating factor receptor (CSF 1R). PGA1, highly expressed in primary stomach carcinoma tissues, is 100%homology to tumor metastasis suppressor gene KAI 1. Conclusions: The improved silver stained RNA AP PCR method is suitable for identification and cloning differentially expressed genes. The results suggest that CSF 1R may be related to the metastatic phenotype of stomach carcinoma.