title: Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.
abstract: DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing.
authors.affiliation: Chronic Viral Diseases Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. MSteinau@cdc.gov
authors.forename: Martin
authors.initials: M
authors.lastname: Steinau
authors.forename: Sonya S
authors.initials: SS
authors.lastname: Patel
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Unger
mesh_terms: D001005:Anus Neoplasms / Q000821:virology; D036022:Capsid Proteins / Q000235:genetics; D002278:Carcinoma in Situ / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D005838:Genotype; D006258:Head and Neck Neoplasms / Q000821:virology; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D018307:Neoplasms, Squamous Cell / Q000821:virology; D009856:Oncogene Proteins, Viral / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016612:Paraffin Embedding; D014565:Urogenital Neoplasms / Q000821:virology
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D005404:Fixatives; D009856:Oncogene Proteins, Viral; D005557:Formaldehyde; C063317:L1 protein, Human papillomavirus type 16