BMC Genomics 2007, 8:117 http://www.biomedcentral.com/1471-2164/8/117 the modeling performance of cell lines (Figure 7B). The "Cell-Cell Signaling" pathway contains 507 genes that transfer information from one cell to another. Some genes in this pathway include fibroblast growth factor, gap junc- tions, interleukins, and leptin precursors. The HeLa gene expression in the "Cell-Cell Signaling" pathway was more similar to both normal cervix and cervical cancer when cultured in the organotypic environment versus monol- ayer as well as the organotypic control. Therefore, HeLa organotypic experiments appear better models of cervical cancer than HeLa cultured in monolayer. The addition of fibroblasts allowed the formation of cell layers, which increased the cell-cell contact. This increase in contact apparently changes the communication between cells and thus increases the consistency in expression patterns of genes involved in cell-cell communication. Conclusion Expression profiles of cervical cancer biopsies were com- pared to previous cervical cancer research to provide evi- dence that the expression profiles accurately represent both cervical cancer and normal cervix. Primary normal cells and the C4-I and C4-II cells lines were found to be better models than the other cell lines we tested, even the more commonly used HeLa cell line. We found that sim- ple changes to the environment, such as media, increased the correlation of HeLa and SiHa cells to cervical cancer. In addition, culturing HeLa and SiHa cell lines in an orga- notypic environment rather than in monolayer signifi- cantly increased their correlation to cervical cancer. The correlation of each cell line and growth condition was also analyzed at the pathway level. Despite the fact that many cell lines still retain a high expression correlation to cervi- cal cancer, our pathway level analysis also revealed cell lines that had an anti-correlation to cervical cancer. Cell lines with low correlations to cervical cancer should be avoided in future studies as models of this disease. Methods Monolayer cell culture Nine cell lines were cultured in monolayer as well as under various perturbations, such as different media and structural environments. All cell lines except the primary normal epithelial line were obtained from the ATCC and cultured in ATCC recommended media. In addition to the recommended media, HeLa and SiHa cells were also grown in DMEM to assess how media changes their corre- lation to cervical cancer. Cell lines were cultured in 10% fetal bovine serum (FBS) (ATCC) and 1:100 PenStrep (Invitrogen). The primary cell line was a gift from Dr. Rebecca Richards-Kortum's lab at Rice University, and was grown in basal media and growth factors (cc-3118, Clonetics). All cell lines were cultured three times inde- pendently across three separate passages except the pri- mary line, which was cultured at the same time in three separate plates due to the cells' short life spans. The cell line media conditions are summarized in Additional file 2. The replicates were individually hybridized to microar- rays. Organotypic cell culture Organotypic cultures consisted of NIH 3T3 fibroblasts, collagen, and an epithelial cell line. Collagen was pre- pared by adding 2.2 ml of type I rat tail collagen (3 mg/ ml) (Roche), 220 µl of 10 × DMEM, 220 µl of FBS, and Hepes-NaOH for a final pH of 7.2. Fibroblasts were resus- pended in prepared collagen at a cell density of 3 × 105/ ml in 3 ml of collagen. 120 µl of the fibroblast/collagen suspension was added to each transwell plate (3 µm pore size and 6.5 mm diameter) (Corning) and incubated for 30 minutes at 37°C to solidify the collagen. SiHa and HeLa cells were resuspended at a concentration of 1 × 106/ ml in DMEM media and 80 µl were added on top of the solid fibroblast/collagen suspension. 600 µl of DMEM media was added to the outside of the transwell insert and was replaced every other day. The organotypic control cul- tures were treated identically to the organotypic cultures, but without adding fibroblasts to the collagen; the absence of fibroblasts prevented 3-dimensional growth of the epithelial cell line. Cervical tissue Three normal and nine moderately to poorly differenti- ated, invasive cervical cancer biopsies were obtained from the Cooperative Human Tissue Network (CHTN) and the Gynecologic Oncology Group (GOG) with the approval of the IRB at The University of Texas at Austin. Two tech- nical replicates from each patient biopsy were hybridized separately to microarrays. mRNA isolation and amplification Total RNA was isolated using Trizol (Invitrogen). RNA quality and quantity was assessed by gel electrophoresis and UV spectroscopy. DNA contamination was removed by RNeasy MinElute Cleanup (Qiagen). Universal Human Reference (UHR) RNA (Stratagene) was used as the refer- ence channel in all hybridizations. RNA was amplified using T7 Message Amp (Ambion). cDNA microarrays Microarrays were printed on poly-L lysine coated slides with 47,000 previously sequence-verified IMAGE clones (Research Genetics/Invitrogen) on each slide, as described by Gu and Iyer [39]. The reference channel consisted of 4 µg of amplified UHR. The experimental samples consisted of 4 µg of amplified RNA from cell lines or biopsies. Amino-allyl labeling and hybridization protocols were performed as previously described [39]. Samples were hybridized for 16 hours in the dark in humidity chambers (Corning). Slides were then washed, dried, coated in Dye- Page 10 of 13 (page number not for citation purposes)