Identification of unique and common low abundance tumor-specific transcripts by suppression subtractive hybridization and oligonucleotide probe array analysis

A54

Most human cancers are characterized by genetic aberrations accompanied by altered expression and function of numerous genes. Applying genome-wide, microarray gene expression analysis to identify deregulated genes in different tumor types can provide potential gene candidates as diagnostic and prognostic tools and promising targets for drug development. However, the detection of genes with low levels of expression remains a major challenge. In this study, we have designed a strategy, termed modified suppression subtractive hybridization (mSSH), to identify genes encoding rare transcripts. The strategy entails incorporating the T₇-promoter sequence at the 5’end of the non-coding cDNA strand during first strand cDNA synthesis so as to generate uni-directional anti-sense RNA from the resultant cDNA following conventional SSH. These transcripts are subsequently analyzed by Affymetrix oligonucleotide gene arrays. Biopsies from five hepatocellular carcinoma (HCC), five breast carcinoma, and four nasopharyngeal carcinoma (NPC) were employed separately as testers and their corresponding normal biopsies as drivers to enrich for low abundance tumor type-specific transcripts. The total detectable number of probe sets following mSSH was reduced by an order of magnitude of almost ten in comparison to those detected for the same tumor resected tissues without undergoing subtraction, thus yielding a subtraction efficacy of over 90%. Using this experimental approach, we have identified 48 HCC-specific, 45 breast carcinoma-specific, and 83 NPC-specific genes. In addition, 115 genes were up-regulated in all the three cancer types. When compared to gene-profiling data obtained without mSSH, the majority of these identified transcripts were of low abundance in the original cancer tissues. mSSH can therefore serve as a comprehensive and unbiased molecular strategy for pursuing functional genomic studies of human cancers.