BMC Genomics 2005, 6:130 http://www.biomedcentral.com/1471-2164/6/130 Micro SAGE protocol Micro SAGE was performed according to Datson et al. [21] with minor modifications, by means of the Invitrogen's I- SAGE kit (Invitrogen, San Diego, CA USA). RNA isolation was done in TRIzol according to manufacturer's instruc- tions. Five µ g of total RNA was used as input material. A heating step was introduced at 65°C for 10 minutes fol- lowed by 2 minutes on ice to allow a better separation of concatenamers [37]. Products greater than 300 bp and smaller than 2,000 bp were excised, extracted and cloned in the SphI site of pZero vector. Clones were selected and screened for inserts by PCR. Cervix library was sequenced by Agencourt through SAGE sequencing service (CGAP collaboration, GR). Sequence files were analyzed with the SAGE300 software [18,20], which identifies the anchoring enzyme sites and extracts the two tags flanked by NlaIII site. Gene identity and UniGene cluster assignment of each SAGE tag was obtained by using the tag-to-gene "reli- able" map, from SAGEmap NCBI site [22,23]. The tags extracted were uploaded to SAGEmap and corresponding accession numbers were retrieved using the H. sapiens NCBI-GenBank database. Reverse Transcription-Polymerase Chain Reaction (RT- PCR) analysis Total RNA was extracted from six normal cervical tissues using TRIzol, quantified by densitometric analysis and its quality evaluated by denaturing gel electrophoresis. Con- taminiating DNA was digested and removed with Rnase- free Dnase (Promega). Expression analysis was performed using 100 ng total RNA in a RT-PCR reaction (Access RT- PCR System, Promega). The mRNA was reverse-tran- scribed at 48°C for 45 min. After an initial denaturation at 94°C for 2 minutes, the double stranded cDNA synthesized was amplified for 40 cycles with denaturation at 94°C for 30 seconds, annealing at 54–60°C for 1 minute and extension at 70°C for 2 minutes with specific oligonucleotides (Table 4) in a Perkin Elmer 480 Thermocycler. Sense and antisense sequence of oligonucleotides for S100 A8 and 9, SPRR3, NICE-3 and -4 genes were designed with the program Primerquest [38]. GAPDH gene expression was used as an internal control. Competing interests The author(s) declare that they have no competing interests. Authors' contributions C.P.P. carried out the microSAGE protocol, real time RT- PCR validations, the bioinformatics analysis and writing the manuscript. G.R. provided sequencing of SAGE library. J.M. helped to write the manuscript and partici- pated in discussions. H.G. A.H. and P.P.S. helped for the bioinformatics analysis and database comparisons. M.S. is the principal investigator and was involved in the concep- tualization, design and writing of the manuscript. All authors read and approved the final manuscript. Additional material Additional data file 1 Tags shared between fibroblast and cervix. Tags founded in SAGE_cervix_normal_B_1(30418 tags) BJ dermal fibroblasts (57573 tags) and SAGE_cervix_normal_B_1 (30418 tags). Ubiquous tags were deleted in both libraries. BJ dermal fibroblast library was derived from neonatal foreskin primary fibroblasts cultured in Ham's F10 medium sup- plemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. Library was developed by Agnes Baross at British Columbia Genome Sciences Centre. Columns are: Gene TAG, Cluster ID and Gene Name Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-6-130-S1.xls] Acknowledgements This work was partially supported by grants of CONACyT (F7114 and 34686-M, MS), and FOFOI-IMSS (MS). For the sequencing of SAGE library, funding was provided by the National Cancer Institute's Cancer Genome Anatomy Project (NIH 23XS073 and 24XS070, GR). During this work CPP, GVO, PPS were recipients of CONACyT, DGEP-UNAM and IMSS fellowships. This work was submitted in partial fulfilment of the requirements for the D. Sc. degree in for PPC at DOCTORADO EN CIENCIAS BIOMEDICAS, UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO. References 1. Mexican Ministry of Health: Registro Histopatológico de Neo- plasias Malignas, Compendio de mortalidad y morbilidad. Secretaría de Salud, México; 1998. 2. Schiffman MH, Bauer HM, Hoover RN, Glass AG, Cadell DM, Rush BB, Scott DR, Sherman ME, Kurman RJ, Wacholder S: Epidemio- logic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasias. J Natl Cancer Inst 1993, 85:958-964. 3. Villa L: Human papillomaviruses and cervical cancer. Adv Can- cer Res 1997, 71:321-341. 4. 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