BMC Genomics 2005, 6:130 http://www.biomedcentral.com/1471-2164/6/130 A number of recent studies about gene expression profiles in in vitro HPV-infected cultured keratinocytes and from (CC) clinical samples have provided an initial notion of the changes in gene expression induced by HPV and in early CC [5-10]. Moreover, some studies have compared normal versus tumor-induced gene expression in cervical samples with the aim to identify potential tumor markers of clinical value [11-13]. At present, there are reports of genes expressed by kerati- nocytes derived from a normal human epidermis and from mouse uterus carried out by Serial Analysis of Gene Expression (SAGE) [14-17]. However, no such study exists for human cervix. Therefore, the aim of our study was to describe the first compendium of expressed genes in nor- mal cervical epithelium, which is composed mainly by keratinocytes strongly influenced by hormones. To achieve this we used SAGE, which is capable of producing an accurate molecular picture of cervical tissue based on expressed genes, as the main methodology. As SAGE is not dependent on preexisting databases of expressed genes, it provides an unbiased view of gene expression profiles within the mRNA populations [18]. SAGE allows the simultaneous quantitative and qualitative analysis of thousands of gene transcripts based on two principles: first, 14 mers are sufficient to uniquely identify 95% of cell transcripts [19]; and second, cloning of these 14 bp tags serially with the insertion of a restriction enzyme rec- ognition sequence as an anchor, the throughput is consid- erably increased. To obtain a catalog of expressed genes and their relative frequencies we performed database analysis to relate each tag to its corresponding gene [20]. As an important drawback of SAGE is that a large amount of messenger RNA (2.5–5 µ g polyA RNA) is required, and our tissue supply was limited (a punch biopsy) we employed the MicroSAGE protocol in RNA thereof [21]. The present report describes a partial transcriptome of a sample derived from normal cervical epithelium used to construct a SAGE library with 30,418 sequenced tags. Results and discussion SAGE library derived from one normal uterine ectocervical sample Our Sage library was obtained from ectocervical tissue from a 38 year old healthy woman with active sexual life, not taking any hormonal therapy, nor any other drug that could potentially alter cervical physiology, we designated this as SAGE_cervix_normal_B_1. Histological analysis of this sample by H&E revealed normal ectocervical tissue, approximately 80% epithelium and 20% stroma without evidence of glands. There were minimal inflammatory infiltrates in the periphery of the sample, considered nor- mal for this type of tissue. The SAGE library yielded 30,418 sequenced tags, which was used to generate a table, which represents genes expressed in normal human cervix. For a complete list of the expressed genes, please visit the SAGEmap website, [22,23]. The derived catalog of expressed genes represents the first attempt to generate a comprehensive and pro- found analysis of the cervical epithelium expression pro- file. The wealth of information obtained allows detection of genes involved in normal epithelium physiology, as well as possible target genes of HPV infection. In general, tag frequency in a typical SAGE experiment follows a nor- mal distribution [24,25]. Table 1 summarizes the general statistics of this library. As seen there is a normal distribu- tion, where only a limited number of tags were either highly expressed or at an extremely low frequency (4.4 and 4.9%, respectively). Tags with a frequency of 1 were not considered for quantitative purposes, because these are likely to represent artifacts of sequencing or of the SAGE procedure [26]. Representativity of the data According to Zhang et al. [27], a study of SAGE data min- ing analysis of 300,000 tags, 75% of mRNA consists of transcripts expressed at more than five copies per cell, and, in general, transcripts are expressed at a range from one to 5,300 copies per cell. With this in mind, our ~30,000-tag library, represents 10% of the total tags analyzed by Vel- culescu et al. The most frequently represented tag in the current report had a frequency of 515 (16,930 tags per million TPM). An estimate of such data indicates that this gene tag has an expression level of ~5,150 copies per cell, similar to what is observed in digital northerns of other top expressed tags in SAGE libraries (Figure 1A). We have to keep in mind, however, that in certain tissues, some genes are expressed at much higher levels, such as growth hormone, with 149,630 TPM in pituitary gland [28]. Because SAGE analysis represents a qualitative and quan- titative assay of messenger RNA abundance not biased by cloning or polymerase chain reaction efficiency [29], our data provide an estimate of the genes normally expressed by normal uterine cervix. Among the most frequently expressed tags in our library (Table 2), some corresponded to ubiquitously expressed transcripts (GRIN2C, FTH1, GNS, RPLP2, RPL21). The presence of this type of genes is a common result in SAGE experiments with an expected heterogeneity in their expression levels [14,15,17,19], indicating a possible role as housekeeping genes (Figure 1B). In a report Velculescu et al., by means of data base analysis of SAGE libraries, found that ~1,000 genes are present in all normal or tumor tissues analyzed with over five copies per cell [30]. Hence, this list of genes identified by data mining is termed minimal transcriptome (i. e., the set of genes expressed by every cell), which represents genes constitu- Page 2 of 9 (page number not for citation purposes)