Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues. 2011

Martin Steinau, and Sonya S Patel, and Elizabeth R Unger
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

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.

UI MeSH Term Description Entries
D009856 Oncogene Proteins, Viral Products of viral oncogenes, most commonly retroviral oncogenes. They usually have transforming and often protein kinase activities. Viral Oncogene Proteins,Viral Transforming Proteins,v-onc Proteins,Transforming Proteins, Viral,v onc Proteins
D002278 Carcinoma in Situ A lesion with cytological characteristics associated with invasive carcinoma but the tumor cells are confined to the epithelium of origin, without invasion of the basement membrane. Carcinoma, Intraepithelial,Carcinoma, Preinvasive,Intraepithelial Neoplasms,Neoplasms, Intraepithelial,Intraepithelial Carcinoma,Intraepithelial Neoplasm,Neoplasm, Intraepithelial,Preinvasive Carcinoma
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D005404 Fixatives Agents employed in the preparation of histologic or pathologic specimens for the purpose of maintaining the existing form and structure of all of the constituent elements. Great numbers of different agents are used; some are also decalcifying and hardening agents. They must quickly kill and coagulate living tissue. Fixative,Pickling Agents,Agents, Pickling
D005557 Formaldehyde A highly reactive aldehyde gas formed by oxidation or incomplete combustion of hydrocarbons. In solution, it has a wide range of uses: in the manufacture of resins and textiles, as a disinfectant, and as a laboratory fixative or preservative. Formaldehyde solution (formalin) is considered a hazardous compound, and its vapor toxic. (From Reynolds, Martindale The Extra Pharmacopoeia, 30th ed, p717) Formalin,Formol,Methanal,Oxomethane
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D006258 Head and Neck Neoplasms Soft tissue tumors or cancer arising from the mucosal surfaces of the LIP; oral cavity; PHARYNX; LARYNX; and cervical esophagus. Other sites included are the NOSE and PARANASAL SINUSES; SALIVARY GLANDS; THYROID GLAND and PARATHYROID GLANDS; and MELANOMA and non-melanoma skin cancers of the head and neck. (from Holland et al., Cancer Medicine, 4th ed, p1651) Cancer of Head and Neck,Head Cancer,Head Neoplasm,Head and Neck Cancer,Head and Neck Neoplasm,Neck Cancer,Neck Neoplasm,Neck Neoplasms,Neoplasms, Upper Aerodigestive Tract,UADT Neoplasm,Upper Aerodigestive Tract Neoplasm,Upper Aerodigestive Tract Neoplasms,Cancer of Head,Cancer of Neck,Cancer of the Head,Cancer of the Head and Neck,Cancer of the Neck,Head Neoplasms,Head, Neck Neoplasms,Neoplasms, Head,Neoplasms, Head and Neck,Neoplasms, Neck,UADT Neoplasms,Cancer, Head,Cancer, Neck,Cancers, Head,Cancers, Neck,Head Cancers,Neck Cancers,Neoplasm, Head,Neoplasm, Neck,Neoplasm, UADT,Neoplasms, UADT
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001005 Anus Neoplasms Tumors or cancer of the ANAL CANAL. Anal Cancer,Cancer of Anus,Anal Neoplasms,Cancer of the Anus,Neoplasms, Anal,Neoplasms, Anus,Anal Cancers,Anal Neoplasm,Anus Cancer,Anus Cancers,Anus Neoplasm,Cancer, Anal,Cancers, Anal,Neoplasm, Anal,Neoplasm, Anus
D014565 Urogenital Neoplasms Tumors or cancer of the UROGENITAL SYSTEM in either the male or the female. Genitourinary Cancer,Genitourinary Neoplasms,Urogenital Cancer,Genito-urinary Cancer,Genito-urinary Neoplasm,Neoplasms, Genitourinary,Neoplasms, Urogenital,Cancer, Genito-urinary,Cancer, Genitourinary,Cancer, Urogenital,Cancers, Genito-urinary,Cancers, Genitourinary,Cancers, Urogenital,Genito urinary Cancer,Genito-urinary Cancers,Genito-urinary Neoplasms,Genitourinary Cancers,Genitourinary Neoplasm,Neoplasm, Genito-urinary,Neoplasm, Genitourinary,Neoplasm, Urogenital,Neoplasms, Genito-urinary,Urogenital Cancers,Urogenital Neoplasm

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