Samples of tumor and normal gastric mucosa were collected from gastrectomy specimens from Queen Mary Hospital, The University of Hong Kong. Tissues were frozen in liquid nitrogen within half an hour after they were resected. Non-neoplastic mucosa from stomach, small intestine and colon was dissected free of muscle, and histologically confirmed to be tumor-free by frozen section. Total RNA was extracted using Trizol (Gibco BRL) and mRNA was isolated by the FastTrack mRNA isolation kit (Invitrogen). This study was approved by the Ethics Committee of the University of Hong Kong and the IRB from Stanford University.
Tumors were classified using the Lauren’s classification into intestinal, diffuse, mixed and indeterminate types (1). The presence of H. pylori in the gastrectomy specimens was determined by histological examination, supplemented by Modified Giemsa staining. The presence of EBV in cancer cells was assayed by in-situ hybridization for EBER as described previously (2). The tumor stage was defined by the General Rules for Gastric Cancer Study of the Japanese Research Society for Gastric Cancer (3).
Microarray procedure and data analysis
We use a cDNA microarray containing 44,500 cDNA clones, representing about 30,300 unique genes. The methods for microarray production and hybridization, and data analysis, including hierarchical clustering, closely followed previously described procedures. (4, 5) In brief, 2 micrograms of sample mRNA and 2 micrograms of reference mRNA were labeled with Cy5-dUTP and Cy3-dUTP (Amersham) respectively, using Reverse Transcriptase (GibcoBRL) for 2 hours at 42o C. The two labeled cDNA probes were separated from unincorporated nucleotides by filtration, mixed and hybridized to microarray at 65o C overnight. After hybridization, each microarray was washed with 2xSSC, 0.03%SDS for 5 minutes at 65o C, then with 1xSSC for 5 minutes and 0.1xSSC for 5 minutes, both at room temperature. The array was then scanned using GenePix 4000A microarray scanner (Axon Instruments). Primary data collection and analysis were carried out using GenePix Pro 3.0 (Axon Instruments). Areas of the array with obvious blemishes were manually flagged and excluded from subsequent analysis. The raw data were deposited into Stanford Microarray Database (6) at: http://genome-www4.stanford.edu/MicroArray/SMD/index.html.
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