BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 Table 2: Functional classes of genes enriched in the RGP library in comparison to the frequency within the whole set of predicted human genes Functional Class (biological process) Genes establishment of cellular localization AP1G1, DYNC1I2, COPZ1, NUP160, KIF5B, RANBP5, PAFAH1B1, FLJ10292, C14orf108, RAN, SET, SGNE1, SSR1, SSR2, BAT1, SEC24C nucleocytoplasmic transport NUP160, RANBP5, FLJ10292, RAN, SET, BAT1 organelle organization and biogenesis ARPC3, DCTN6, MYST2, DDX1, DYNC1I2, XRN2, KIFAP3, DAAM1, POT1, H3F3A, HDAC1, HMGB2, KIF5B, STMN1, PAFAH1B1, ATRX, KLHL4, PXMP3, RAN, SET, SMYD3, WASPIP, ACTL6A, H2AFV, chromosome organization and biogenesis MYST2, POT1, H3F3A, HDAC1, HMGB2, ATRX, SET, SMYD3, ACTL6A, H2AFV microtubule-based process DYNC1I2, XRN2, KIFAP3, KIF5B, STMN1, PAFAH1B1, RAN nucleobase biosynthesis PAICS, PPAT regulation of protein biosynthesis DDX1, EIF4B, EIF4G2, PUM2, TLR3, EIF4E2 DNA metabolism POLD3, MYST2, XRN2, POT1, H3F3A, HDAC1, HMGB2, NONO, ORC2L, ATRX, RAD23B, RAN, SET, SMYD3, UBE2A, XRCC5, HAT1, ACTL6A, H2AFV DNA packaging MYST2, H3F3A, HDAC1, HMGB2, SET, SMYD3, HAT1, ACTL6A, H2AFV DNA repair POLD3, XRN2, HMGB2, NONO, ATRX, RAD23B, UBE2A, XRCC5 response to DNA damage stimulus POLD3, XRN2, HMGB2, NONO, ZAK, ATRX, RAD23B, UBE2A, XRCC5 RNA metabolism SYNCRIP, DDX17, SF3A3, DDX1, DCP2, ELAVL1, XRN2, SF3B1, LSM5, HNRPC, HNRPU, NONO, FLJ10292, RARSL, SNRPG, BAT1, TTF2, SIP1, DDX23 RNA processing SYNCRIP, DDX17, SF3A3, DDX1, XRN2, SF3B1, LSM5, HNRPC, HNRPU, NONO, FLJ10292, SNRPG, BAT1, TTF2, SIP1, DDX23 RNA splicing SYNCRIP, SF3A3, DDX1, SF3B1, LSM5, HNRPC, NONO, FLJ10292, SNRPG, BAT1, TTF2, SIP1, DDX23 RNA localization NUP160, FLJ10292, RAN, BAT1 macromolecule catabolism YME1L1, DDX1, DCP2, ELAVL1, XRN2, USP33, ARIH1, MDH1, PSMA4, PSMA5, PSMB4, PSMB6, UBE2A, USP8 Relative enrichment significance O = 16a; E = 8.04b; R = 1.99c P = 0.0067d O = 6; E = 1.61; R = 3.73; P = 0.0054 O = 24; E = 10.92; R = 2.2; P = 0.00022 O = 10; E = 4.05; R = 2.47; P = 0.0076 O = 7; E = 2.13; R = 3.29; P = 0.0056 O = 2; E = 0.12; R = 16.67; P = 0.0064 O = 6; E = 1.66; R = 3.61; P = 0.0064 O = 19; E = 8.44; R = 2.25; P = 0.00078 O = 9; E = 3.42; R = 2.63; P = 0.0074 O = 8; E = 2.91; R = 2.75; P = 0.0088 O = 9; E = 3.24; R = 2.78; P = 0.0053 O = 19; E = 5.8; R = 3.28; P = 5.44-06 O = 16; E = 4.68; R = 3.42; P = 1.84374398023E-05 O = 13; E = 2.01; R = 6.47; P = 1.03E-07 O = 4; E = 0.64; R = 6.25; P = 0.0037 O = 14; E = 4.57; R = 3.06 P = 0.00019 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 Analysis of the expression profile of the genes represented in the SSH libraries in a panel of melanocytic samples using a publicly available microarray study Since our validation results indicate that we have two col- lections of genes with truly differential expression between an RGP and a pool of metastatic melanoma cell lines, we decided to analyze the behavior of the subtracted genes in a panel of human melanoma tumors. We searched the data tables of a publicly available microarray study [16] that contains the expression profiles of samples from normal skin (3 samples), nevi (9 samples), primary (6 samples) and metastatic (19 samples) melanomas hybridized against a cDNA microarray containing 20,862 human cDNAs (representing 19,740 unique genes) from Research Genetics (Huntsville, AL). This work showed that metastatic melanomas exhibit two different gene expression signatures and that one of these signatures is shared with a sample from an RGP melanoma lesion. We were able to extract expression data for 194 genes of the RGP library and for 155 genes of the Met library. First, the set of data for each group was submitted to SAM (Signifi- cance Analysis of Microarray), setting the False Discovery Rate (FDR) to zero, in a two-class unpaired analysis where one group was represented by primary melanomas and the other group by metastatic melanomas. As shown in Fig. 5, a subset of RGP library genes was able to distin- guish primary melanomas from metastasis, although two metastatic samples grouped with the primary tumors. A small group of genes from the RGP library were pointed as differentially expressed between primary and metastatic melanomas, including genes overexpressed in primary tumors as well as genes overexpressed in metastatic melanomas. The genes detected as down-regulated in most metastatic melanomas, and that are therefore candi- dates for metastasis suppressors, are LUM (lumican), DCTN6 (dynactin 6) and DNCI2 (dynein intermediate chain 2) (Fig. 5A). The Met library genes were not able to distinguish between primary and metastatic melanomas, since only one gene, ENDOD1 that codes for a putative endonuclease, was detected by SAM, at FDR = 0, as differ- entially expressed between the two tumor stages. In a sec- ond analysis, we performed SAM to compare non- Page 9 of 18 (page number not for citation purposes)