BMC Genomics 2005, 6:130 Table 1: SAGE data general statistics. Frequency distribution a >200 tags 100–200 tags 20–100 tags 5–19 tags 2 – 4 tags 1 tag Total No of unique tags Tags with SAGE database matches No. of different transcripts matched No of poorly characterized transcripts Genes with known functione tively expressed. In supplementary information of Velcu- lescu's work [30] a search for the minimal transcriptome in our library, indicates >95% of housekeeping genes (data not shown), further validating the cervical library. Spectrum of genes expressed by normal cervical tissue To obtain better knowledge of the functional categories of global gene expression profile, we employed the Fatigo Data mining website [31,32]. Figure 2 shows the distribu- tion of expressed genes by functional categories defined by the Gene Ontology Consortium. As seen, the most fre- quent individual transcripts correspond to genes involved in maintenance and basic metabolism. On the other hand, genes corresponding to other processes such as cell growth regulation, morphogenesis, cell differentiation, or death were not as frequently expressed. Top expressed non-ubiquitous genes in normal cervical tissue mainly correspond to epithelial growth and differentiation It was important to distinguish which non-ubiquitous genes were predominantly expressed in normal cervix. As seen in table 2, genes related with epithelial differentia- tion and squamous architectural maintenance are abun- dantly represented in our library. These include S100A8, S100A9, and SPRR3, that belong to a complex of genes that are subject to coordinate regulation during keratino- cyte differentiation. This complex has been called the epi- dermal differentiation complex (EDC) and is located on the 1q21 chromosome [33,34]. These genes share spatial and temporal expression and interrelated functions and aCalculation of the frequency distributon of a given tag was based on total tags sequenced in library. bSome genes have more than one tag, hence we search for individual genes in a database created in SQL language. c,dPercentage of tags or genes in frequency group. ebased on FatiGO Datamining website [31] Tags 1,348 (4.4%) 2,371 (7.8%)c 5,536 (18.19%) 6,349 (20.8%) 13,316 (43.7%) 1,498 (4.9%) 30,418 (100%) http://www.biomedcentral.com/1471-2164/6/130 8,062 5,255 4,340 1,215 2,453 Individual Genesb 5 (0.11%) 21 (0.5%)d 80 (1.8%) 575 (13.2%) 3,050 (70.4%) 609 (14.1%) 4,340 (100%) are grouped in three related gene families: cornified enve- lope precursor proteins (involucrin, loricrin, and the small proline-rich proteins [SPRRs]); intermediate fila- ment-associated proteins (profilaggrin and trichohyalin), and calcium binding proteins (the S100As) [reviewed in [35]]. Approximately 30 genes belonging to the EDC are clustered together in a 200 Mb region, from which there are 20 genes expressed the in cervical SAGE library (Table 3). End point RT-PCR analysis confirms expression of genes detected by SAGE It was important to confirm the expression of some EDC representative genes in different normal cervical tissues by a different technique. For this, we chose end point reverse transcriptase polymerase chain reaction (RT-PCR) analy- sis. Figure 3 shows the expression of five EDC genes in HPV negative tissue samples with no histopathologic lesion. As expected, the majority of cases expressed these genes. However, there were some differences in the level of expression among the different normal samples. This could be due to the fact that samples were taken on differ- ent days of the menstrual cycle (hormonal influence) or to unknown physiological differences among biological systems. Minor expression of fibroblast-related genes in cervical tissue The gene expression catalog reported on here was obtained from a heterogeneous population of cells composed mainly of epithelial keratinocytes in dissimilar Page 3 of 9 (page number not for citation purposes)