Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues

• Autores: Martin Steinau, Sonya S. Patel, Elizabeth R. Unger
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 13, Nº. 4, 2011, págs. 377-381
• Idioma: inglés
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• Resumen

  • 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-\u03bcm 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.