To validate the results obtained from the SSH/microarray assays, we randomly selected six differentially expressed genes identified only by the SSH/microarray assays and three differentially expressed genes identified by both the regular cDNA microarray and SSH/microarray assays for the comparison of gene expression between hepatoma and the corresponding non-hepatoma liver tissues in eighteen patients of HCC using reverse transcription realtime PCR (qRT-PCR). Total RNA was extracted from tissues with Trizol reagents and reverse transcribed using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA). qRT-PCR was conducted using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Pre-designed Assays on Demand TaqMan probes and primer pairs for these 9 genes were obtained from Applied Biosystems Incoporated (ABI) (Foster City, CA). For each gene, two to four sets of Taq-Man probes and primers were tested. The probes contained a 6-carboxyfluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end. These were designed to hybridize across exon junctions. As a result, no fluorescent signal was generated by these assays when genomic DNA was used as a substrate, which confirmed that the assays measured only mRNA. Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate, and 18S ribosomal RNAs were used as internal controls.