BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 cSFeuiglblsutrraect2ed cDNA profiles of the RGP and metastatic (Met) Subtracted cDNA profiles of the RGP and metastatic (Met) cells. PCR-1 represents the PCR products generated using a single primer directed towards both adaptors, after 27 amplification cycles from two duplicate samples of sub- tracted (S1 and S2) or non-subtracted (NS) cDNA of the RGP (WM1552C) and the metastatic (a pool of WM9, WM852, 1205Lu and WM1617) cell lines. PCR-2 represents the PCR product generated after 10 amplification cycles by nested-PCR using a specific primer for each adaptor. Note the difference between the subtracted and non-subtracted profiles. redundancy of 59%). Additional File 2 lists the identifier and annotation of the genes represented in the RGP (Tables S1) and Met (Table S2) libraries and indicates the number of sequence occurrences for each gene in the library. Common to both libraries, there were 10 genes represented by 22 sequences, which corresponded to only 2.5% of the total number of sequences analyzed (Table 1, and Additional File 2). A total number of 24 and 19 sequences from the RGP and Met libraries, respectively, match only EST (Expressed Sequence Tag) sequences in the GenBank, thus suggesting that the libraries may con- SSH collections Number of clones obtained Sequences analyzed Sequences matching human mRNAs/ESTs Sequences matching introns Chimerical clones Sequences matching intergenic regions Sequences matching mitochondrial genome Number of genes represented Genes represented by more than one clone Sequences corresponding to genes common in both libraries Table 1: Global analysis of the clones generated by Suppression Subtractive Hybridization tain sequences representing rare human transcripts. In addition, 8 RGP sequences and 5 Met sequences mapping to the human genome sequence (Additional File 3, Fig. S2–S14) do not match any expressed sequences and thus they might represent novel transcripts. Even among the sequences matching known human mRNAs we obtain additional information. For example, a sequence from the Met library aligns to and extends a putative alternative exon of a cDNA corresponding to the gene ABCB5 (Addi- tional File 3, Fig. S15). Assuming that the genes represented by multiple clones within each library are the ones with the highest differen- tial expression levels between the RGP and metastatic cell lines, we reviewed the literature on these genes by search- ing for their involvement in cancer in general and specifi- cally in melanoma, as summarized in the Additional File 4 (Tables S3 and S4). In the RGP library, 37 genes are rep- resented by at least 2 sequences (maximum number of clones for a gene is 10) and in the Met library this number is 32 genes (maximum number of clones for a gene is 55). Among these 37 genes from the RGP library, 18 have been reported with some alteration in cancer (only 3 of them in melanoma) whereas the 19 remaining have not been associated to cancer. In the Met library, 23 from the 32 genes have been associated to cancer, including 10 also associated to melanoma development. Validation of the expression pattern of genes identified in the subtractive libraries The identification of only 2% of the clones shared by both libraries strongly suggested that the cDNA subtraction was highly efficient. However, to confirm that this was indeed the case, we selected 7 genes for validation by Northern blots and RT-PCR in a panel of 6–8 melanoma cell lines that represent the three stages of tumor progression, including the cell lines used for the SSH libraries (Fig. 3). The genes selected for validation from the RGP library were DCN (represented by 8 clones), ALS2CR7 (10 clones) and MBOAT1 (3 clones). DCN encodes decorin, a secreted protein involved in cell growth regulation and RGP library 2016 367 (18.5%) 351 (94.1%) 5 (1.3%) 2 (0.5%) 4 (1.1%) 5 (1.3%) 288 (82%) 37 (11%) 12 (2.2%) Met library 1920 386 (20.5%) 368 (95%) 1 (0.26%) 2 (0.5%) 4 (1.0%) 11 (2.8%) 217 (59%) 32 (8.7%) 10 (2.5%) Page 6 of 18 (page number not for citation purposes)