Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

Baldwin, Don A.; Feldman, Michael; Alwine, James C.; Robertson, Erle S.

Oropharyngeal squamous cell carcinoma tumor samples were obtained from the Abramson Cancer Center's Tumor Tissue and Biospecimen Bank (https://somapps.med.upenn.edu/pbr/portal/tumor/). All samples were reviewed by our resident pathologist for case history and confirmed for tumor type and demarcation of the cancer cells. If significant adjacent normal tissue was present, sections were mounted on noncharged glass slides for dissection of tumor tissue using a template slide with a hematoxylin-and-eosin (H&E)-stained section with the cancer region clearly demarcated. Specimens containing mostly cancer cells were provided as paraffin rolls. The rolls or mounted sections (minimum of 5; 10 m each) from formalin-fixed, paraffin-embedded (FFPE) tumors were used for sequential DNA and RNA extraction using the AllPrep DN A/RNA FFPE kit (Qiagen, Germantown, MD, USA). Nucleic acid quality control assessments included A 260/280 ratios, yield, and size distribution by agarose gel electrophoresis in 0.5Ï« Tris-borate EDTA buffering system. As expected, formalin-exposed RNA was partially degraded. However, recovery of most samples was relatively good, allowing for further processing. In most of the samples, the fragment sizes were acceptable and were moved ahead for cDNA conversion.