Eight archival formalin-fixed, paraffin-embedded primary skeletal Ewing's sarcoma samples from children (age range between 4-17 years) were obtained from Pathology Department, University of Çukurova, in Turkey. Sections (5 µm) were cut from tumor blocks and stained with hematoxylin and eosin to ensure the histological representation of the sample and cross detecting of areas of tumor cells. Based on microscopic evaluation, areas of interest were identified, scraped with a scalpel, collected in sterile eppendorf tubes containing 50 µl DOP-PCR reaction solution in order to amplify the DNA directly.
CGH experiments and the evaluation of the results were performed as described previously [10-12,15] with minor modifications. Briefly, tumor DNA and normal male or female reference DNA (from normal male and female DNA) were labeled with spectrum green-dUTP and spectrum red-dUTP (Vysis Co.), respectively by applying DOP-PCR.
Metaphase spreads were prepared from phytohemagglutinin stimulated lymphocytes of healthy male (46,XY) and female (46,XX) by standard procedures of colcemide arrest, hypotonic treatment and 3:1 methanol/glacial acetic acid fixation.
For CGH, 500 ng of tumor DNA, 500 ng of normal DNA and 50 µg unlabeled Cot-1 DNA were co-precipitated and redissolved in 15 µl hybridization buffer. DNA and metaphase spreads were denatured at 70°C and at 68°C, respectively.
Hybridization was allowed to proceed for 2 days. Post-hybridization washes were carried out to a stringency of 50% formamide/2xSSC at 45°C. Imaging processing was carried out using Mac Probe Program version 3.4 software. Average green:red fluorescence intensity ratio profiles were calculated for each chromosome in 10 metaphases. Defining the gain and loss in DNA sequence copy number in tumors were based on comparison of normal DNAs labeled with two different colors according to previously described protocol [16,17]. The decision limits of the green-to-red ratios were <0.85 for the loss of DNA copy number and >1.40 for the gain of DNA copy number. High level increases were distinguished from low level increase by a cut-off level of 2.0. Also, for DOP-PCR and CGH, various negative and positive control experiments were carried out by crossing different labeled test and control DNAs as well as hybridization on chromosomes.