Qiaamp dna mini extraction kit

DNA was extracted during tumor sample protein extraction protocol for either ER/PR or uPA PAI quantification. Briefly, each frozen tumor specimen was pulverized in liquid nitrogen and homogenized in a Polytron homogenizer (Glen Mills, NJ, USA) with a cytosol extraction buffer (20 mM Tris∙HCI, 1.5 mM ethylenediaminetetraacetic acid [EDTA], 10 mM Na2MoO4, 1.5 mM dithiothreitol and 10 % glycerol, pH 7.4; in the case of tumors processed for ER and PR testing using the DCC assay) or with Triton X-100 buffer (in the case of tumors processed for uPA/PAI-1 testing using the Femtelle kit, American Dignostica) with a buffer:tissue ratio of 10:1 (volume/weight) and centrifuged at 10,000 × g for 15 min. Total genomic DNA was extracted from the pellets obtained by centrifugation of either cytosol or Triton extract using the QIAamp DNA extraction minikit (ref 51304, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Supernatants were used to prepare cytosol or Triton X-100 protein extracts and the total protein content was quantified using the Pierce assay (BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL) as previously described [23 (link)].

DNA was extracted by using the QIAamp DNA extraction Mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNA was used immediately or stored at—20°C until used.