Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE)

Abstract

Background: Serial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC).

Results: We report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation.

Conclusion: This first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Figure 1

A) Comparison of most expressed tags among different SAGE libraries. Normalized expression levels (TPM) are similar between libraries with different total sequenced tags, indicating comparable messenger abundance among top expressed genes. Expression levels were obtained from SAGEmap website . ^aTPM: Tags per million. Normalization to compare libraries with different numbers of sequenced tags. TPM is obtained by the following formula [(Tag frequency)(1000,000)/Total No. of sequenced tags]. ^bDN: digital northern, indicating gene expression level for a specific gene in a library. B. Graphical representation of expression levels (TPM) for three constitutive genes in several normal (N) and tumoral (T) tissues. Brain tissue libraries: SAGE BB542 whitematter (N) and SAGE Brain medulloblastoma B 98 04 P117 (T). Breast: SAGE Breast normal organoid (N) and B SAGE Breast carcinoma epithelium AP DCIS6 (T). Gastric: SAGE normal gastric body epithelial (N) and SAGE Hiroshima GC W246T (T). Liver: SAGE normal liver (N) and SAGE Liver cholangiocarcinoma B K2D (T). Kidney: SAGE Duke Kidney (N) and SAGE_Kidney_carcinoma_B_D2 (T). Colon: SAGE NC2 (N) and SAGE Tu98 (T). Prostate: SAGE PR317 normal prostate (N) SAGE PR317 prostate tumor (T). Lung: SAGE normal lung (N) and SAGE Lung adenocarcinoma MD L10 (T). Expression levels are indicated as tags per million.

Figure 2

Functional categories assigned to individual genes identified in normal cervical SAGE library. Genes can be assigned in different functional categories. ^aThe percentage was calculated with 3,764 initial genes from which 2,720 genes had Gene Ontology classification.

Figure 3

Expression of genes clustered in 1q21, in normal cervical tissues. One hundred nanograms of total RNA purified of each sample was used in one RT-PCR reaction with gene specific primers; then one tenth of each RT-PCR reaction was subjected to agarose gel electrophoresis. MW: molecular weight marker; C1–C6 six different normal cervical samples