Ethical clearance was obtained from the study base, Mariano Marcos Memorial Hospital and Medical Center (MMMH-MC) in Ilocos Norte, Philippines. All participants gave their written informed consent. Formalin fixed paraffin embedded (FFPE) or fresh frozen biopsies from patients with histologically confirmed primary tumors of the head and neck seen at MMMH-MC between January 2003 to September 2013 were utilized in this study. Tissue sectioning, assessment of tumor content, and molecular analyses were performed at the German Cancer Research Center in Heidelberg, Germany.
FFPE and fresh frozen tissue sectioning was performed as described [12 (link), 13 (link)]. Genomic DNA from the fresh frozen sections was extracted by MagNA Pure 96 DNA and viral NA Large Volume Kit (Roche, Penzberg, Germany) following the manufacturer’s recommendations. DNA extraction from FFPE sections was done as described [13 (link), 14 (link)]. Total RNA was isolated from the fresh frozen and FFPE sections using the RNeasy Minikit (Qiagen, Hilden, Germany) and Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA), respectively. DNAse I digestion was performed prior to the last washing step to ensure exclusive amplification of RNA [14 (link)].
FFPE and fresh frozen tissue sectioning was performed as described [12 (link), 13 (link)]. Genomic DNA from the fresh frozen sections was extracted by MagNA Pure 96 DNA and viral NA Large Volume Kit (Roche, Penzberg, Germany) following the manufacturer’s recommendations. DNA extraction from FFPE sections was done as described [13 (link), 14 (link)]. Total RNA was isolated from the fresh frozen and FFPE sections using the RNeasy Minikit (Qiagen, Hilden, Germany) and Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA), respectively. DNAse I digestion was performed prior to the last washing step to ensure exclusive amplification of RNA [14 (link)].
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