The SSH/cDNA microarray versus regular cDNA microarray approaches To conduct the comparative transcriptomic studies on human hepatoma particularly for the genes at low-expres-sion levels, we set out an approach as shown in Figure 1 . We used the PCR-based suppressive subtractive hybridization to enrich the differentially expressed cDNA clones between human hepatoma and non-hepatoma liver tissues. The SSH was conducted both forwards using RNA prepared from hepatoma and non-hepatoma liver tissues as tester and driver, respectively, to yield the hepatoma (T-N) subtracted amplicons, and reversely using the mRNA prepared from non-hepatoma and hepatoma liver tissues as tester and driver respectively to yield the non-hepatoma (N-T) subtracted amplicons as well. Instead of the previously reported combination with cDNA microarray approaches, in which clones in the subtracted cDNA libraries were cloned and then printed on chips, we directly labeled the subtracted T-N and N-T amplicons and then used as targets to hybridize the pre-made microarray chips printed with the known 14,811 cDNA clones. We named this modified approach as "SSH/microarray". The same tissue RNA pairs and cDNA microarray chips were also used for the regular cDNA microarray assays for comparison. Since human hepatoma generally occurs in patients of chronic hepatitis with/without cirrhosis, to identify the genes implicated in the common biological processes leading to hepatocellular carcinogenesis, we selected three hepatoma patients with different underlying liver disease. The first was with chronic hepatitis C and cirrhosis, the second had chronic hepatitis B and cirrhosis, and the third had chronic hepatitis B but no clinical or histological evidence of cirrhosis. Figure 2 demonstrates the representative results obtained from the corresponding regular cDNA microarray and the SSH/microarray assays. Of note, in the SSH/microarray assays the genes in the subtracted T-N and N-T amplicons were readily identified without tremendous sequencing works, which were required for the original SSH and microarray combination assays. In addition, many of the clones, including those of the house-keeping genes, such as β-actin and GAPDH, with unremarkable difference in hybridization intensities by the regular cDNA microarray assays were efficiently excluded from the subtracted amplicons ( Figure 2 , indicated by white circles). This indicated the efficiency of subtraction hybridization. Most importantly, many of the clones with hybridization intensity below the evaluation threshold by the regular cDNA microarray approaches (hybridization intensities lower than or close to the noise) became detectable by the SSH/ microarray method ( Figure 2 , indicated with red circles). These observations suggested a more sensitive detection of the low abundance transcripts using the SSH/microarray assays. Figure 1 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. 