Experimental flowchart. Messenger RNA was prepared from hepatoma (T) and the corresponding non-hepatoma liver tissues (N), and then subjected to 1) PCR-based suppressive subtractive hybridization (SSH) followed by using the resulted subtracted cDNA libraries as targets for cDNA microarray analysis (the SSH/microarray), and 2) conventional cDNA microarray analysis. SSH was performed in both the forward (T as tester) and reverse (N as tester) direction to enrich up-regulated (T-N amplicon) as well as down-regulated transcriptomes (N-T amplicon) in human hepatoma, respectively. The two subtracted amplicons were labeled with fluorescent cy-dyes as targets for microarray analysis, in which dye-swapping approaches were used. The results thus obtained were then compared to those obtained from the conventional cDNA microarray assays. The differentially expressed genes were categorized into three groups: I) only detected by conventional cDNA microarray approaches, II) identified by both conventional cDNA microarray and SSH/microarray approaches, and III) only obtained from SSH/microarray assays. The results were further confirmed by qRT-PCR in 6 genes randomly selected from group III differentially expressed genes.