S. Noori, A. Monabati, A.A. Ghaderi association of HPV DNA with ESCCs. Morgan and colleagues used PCR to examine frozen tissue from 22 cases of ESCCs for the presence of spe- cific DNA sequences from oncogenic strains of HPV. The products of PCR were further analyzed by southern blot hybridization (SBH). No HPV sequences were detected in any tumor, suggesting it is unlikely, therefore, that HPV plays a significant role in the pathogenesis of ESCCs in the United Kingdom.17 Saegusa and colleagues examined 103 esoph- ageal carcinomas by PCR method using two consensus (targeting either the L1 or the E6-E7 regions) and two type specific (type 16 and type 18) primer sets. However, the entire series of tumor DNA were negative for HPV sequences by PCR assays using all four primer sets.18 This study was designed to evaluate preva- lence of HPV in ESCC cases diagnosed in Pathol- ogy Department, Medical School, Shiraz Univer- sity of Medical Sciences. Materials and Methods All cases of ESCC that were reported between years 1982 to 2002 in the Pathology Department, Medical School were identified. All slides of ESCC cases (n=92) available in the departmental archive were reviewed, and the best slides and their paraffin- embedded tissue blocks were extracted. In addi- tion, slides and paraffin-embedded tissue blocks of normal esophagus from 20 autopsy cases (15-80 years), who referred to the Department between 1996 to 2000, were extracted. To prepare DNA sample from each block one section for hema- tyoxyllin eosin (H&E) staining and 15 sections for DNA extraction were prepared. All sections had a thickness of 5 µm. The sections for DNA extraction placed in two microfuge tubes. Another section was prepared for H&E staining, and was used to confirm the presence of tumor tissue in all previous sec- tions taken for DNA extraction. In order to prevent cross contamination and pick up of sectioned tissues from previous blocks, blade of microtome, working instruments, and surfaces were cleaned by Xylol and HCL 1N before starting with another block. The extraction of DNA was done by proteinase K incubation technique followed by phenol-chloroform extraction of DNA. Analysis of Extracted DNA Samples Samples of DNA were quantitatively assayed using spectrophotometery by measuring absor- bance at 260 nm (A260), 280 nm (A280), and 230 nm (A230) to determine concentration of ds DNA, and to measure A260 to A280 ratio (optimal ratio is equal to 1.8 or more and less than 2) and A260 to A230 ratio (optimal ratio is greater than 128 2.2). A280 is an estimate of concentration of non- DNA components such as proteins, whereas A230 is measured to estimate concentration of other components such as detergents, peptides, carbo- hydrates, phenol and chloroform. Some randomly selected samples were assayed quantitatively by running on a 2% agarose gel electrophoresis, allowing the molecular weight of the DNA to be estimated. The electrophoresis resulted in a thick smear of DNA fragments with variable size rang- ing from very high molecular weight DNA to those about 100 bp in size (figure 1). Moreover, some of the samples were assayed in a PCR program using primers to amplify cytotoxic T lymphocyte antigen 4 (CTLA4) gene with a product of 162 bp in size that showed sharp specific bands on aga- rose gel electrophoresis indicating good quality of extracted DNA. Because, optimal concentration for PCR is about 0.1- 0.5 µg/ µl, we used 10 µl of stock DNA sample to make optimal concentration equal to 0.3 µg/µl by adding appropriate amount of deionized water to it. The DNA samples were stored in -20°C for later use. Figure 1: Photograph (negative version) of ethidium bro- mide-stained agarose gel after electrophoresis of extracted DNA from some randomly selected cases. (5 µl of stock DNA mixed with 4µl of loading dye applied for electrophoresis un- der 70 volts for 6 hours) Positive Controls Human papilloma virus-18 full length genome, cloned in pBR322 (ATTC 45152), was extracted from transformed E. Coli using the technique of alkaline lysis / phenolic extraction of DNA. It was used as a positive control in each run of PCR assay using general HPV or HPV-18 primer sets. Iran J Med Sci June 2012; Vol 37 No 2