BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 Table 3: Functional classes of genes enriched in the Met library in comparison to the frequency within the whole set of predicted human genes Functional Class (biological process) Genes cell adhesion ADAM10, CTGF, CTNNB1, CTNND1, FN1, ITGA6, ITGB1, ITGB8, LAMA4, NRCAM, SPP1, TGFBI, THBS2, TNFAIP6, HMCN1, CD164, NRP2, NRXN3, CD36 cell-matrix adhesion ITGA6, ITGB1, ITGB8, SPP1 regulation of cell adhesion ADAM10, LAMA4, TGFBI, CD164 integrin-mediated signaling pathway ADAM10, ITGA6, ITGB1, ITGB8 intracellular receptor-mediated signaling pathway CTNNB1, EDD1, RB1, NCOA4 cell differentiation ACVR1C, DCT, GPM6B, MGP, MITF, NRCAM, SERPINE2, SFRP1, SPP1, TYR, TYRP1, NRP2, NRXN3 cell motility CTGF, FN1, ITGB1, LAMA4, NRCAM, SERPINE2, SPP1, NRP2, NRXN3 cell migration FN1, ITGB1, LAMA4, NRCAM, SERPINE2, SPP1, NRP2, NRXN3 nucleocytoplasmic transport ADAM10, KPNA1, NPM1, IPO9, G3BP2, THOC1 negative regulation of cell proliferation GPNMB, FABP7, IL6, NPM1, CUL5, CD164 aromatic amino acid family metabolism DCT, TDO2, TYR, TYRP1 aromatic compound metabolism CPM, DCT, TDO2, TYR, TYRP1 cofactor biosynthesis PBEF1, TMEM131, TPK1, ATP5A1, ATP6V1B2 coenzyme biosynthesis PBEF1, TMEM131, TPK1, ATP5A1, ATP6V1B2 negative regulation of transcription HMGB1, TRIM33, HBXAP, NKRF, RB1, ARID5B positive regulation of transcription CTNNB1, ILF2, NFATC2, HBXAP, RB1, NCOA4 mRNA processing DHX8, PABPC1, GRSF1, SFRS2, SNRPB2, SNRPG, G3BP2, THOC1 pigment metabolism DCT, TYR, TYRP1 Relative enrichment significance O = 19a; E = 6.7b; R = 2.84c; P = 3.71E-05d O = 4; E = 0.6; R = 6.67; P = 0.0030 O = 4; E = 0.4; R = 10; P = 0.00064 O = 4; E = 0.57; R = 7.02; P = 0.0025 O = 4; E = 0.47; R = 8.51; P = 0.0012 O = 13; E = 5.34; R = 2.43; P = 0.0027 O = 9; E = 2.42; R = 3.72; P = 0.00072 O = 8; E = 1.04; R = 7.69; P = 9.41E-06 O = 6; E = 1.25; R = 4.8; P = 0.0016 O = 6; E = 1.59; R = 3.77; P = 0.0052 O = 4; E = 0.23; R = 17.39; P = 6.642E-05 O = 5; E = 0.97; R = 5.15; P = 0.0028 O = 5; E = 1.18; R = 4.24; P = 0.0066 O = 5; E = 1.04; R = 4.81; P = 0.0039 O = 6; E = 1.72; R = 3.49; P = 0.0076 O = 6; E = 1.19; R = 5.04; P = 0.0012 O = 8; E = 2.13; R = 3.76; P = 0.0013 O = 3; E = 0.25; R = 12; P = 0.0018 a: observed number of genes in the category; b: expected number of genes in the category; c: observed/expected ratio; d: p-value of the enrichment significance neoplastic samples (skin and nevi) against tumor samples (primary and metastatic melanomas) for each set of genes (Fig. 5B and 5C). Genes from both libraries were found differentially expressed between non-neoplastic tissues and melanoma in both directions (up-regulated in non- neoplastic tissue/down-regulated in tumor, and down- regulated in non-neoplastic tissue/up-regulated in tumor). Since the WM1552C cells display markers of a less aggressive phenotype, for the RGP gene collection, we indicate a group of 14 genes (Fig. 5B, left side blue line) that were found to be consistently down-regulated in tumor samples. In contrast, a set of 14 Met library genes (Fig. 5C, left side blue line) showed a consistent up-regu- lation in tumor samples, compatible with an oncogenic role. The consistent differential expression profiles of these two groups of genes, distinguishing non-neoplastic from neoplastic tissues and primary melanomas from metastatic ones, make them good candidates for further studies in melanoma. Treeview analysis of the genes validated by Northern blots showed that DCN is preferentially overexpressed in most skin and nevi samples and in 2 (out of 5) primary melano- mas, confirming that its expression seems to decrease with melanoma progression, although a subset of melanoma samples also presented an overexpression of this gene (Fig. 5C, bottom). MITF, which was identified in the Met library, showed down-regulation in all skin samples, an increased expression in most nevi and overexpression in 11 out of 25 samples of primary and metastatic melano- mas (Fig. 5C, bottom). The other two genes from the Met library (PLP-1 and HLA-DRA) did not exhibit expression pattern clearly associated to any specific stage of the melanoma progression in this study (Fig. 5C, bottom). Discussion We report here the generation and analysis of two collec- tions of subtracted cDNAs corresponding mostly to anno- tated mRNA. The collections are unique with only a few of the clones (2.5%) being common to both libraries, indi- cating that these collections represent a transcriptional content that distinguish between the RGP cells WM1552C and a group of four metastatic cell lines, and may reflect distinct transcriptional profiles of these two stages of melanoma progression (Additional File 2, Tables S1 and S2). Some of the sequences identified also contribute to extend annotated mRNA, as is the case for ABCB5 (Fig. S15), or reveal novel transcripts, since they match only genomic DNA, mapping in introns or intergenic regions (Additional File 3, Fig. S2–S14). We further assured that the subtraction process was efficient and validated the libraries by showing that all genes selected for validation Page 10 of 18 (page number not for citation purposes)