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| Content Provider | PubMed Central |
|---|---|
| Author | Steinau, Martin Patel, Sonya S. Unger, Elizabeth R. |
| 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. |
| Related Links | http://dx.doi.org/10.1016/j.jmoldx.2011.03.007 |
| Starting Page | 377 |
| File Format | |
| ISSN | 19437811 |
| e-ISSN | 19437811 |
| Journal | The Journal of Molecular Diagnostics : JMD |
| Issue Number | 4 |
| Volume Number | 13 |
| Language | English |
| Publisher | American Society for Investigative Pathology |
| Publisher Date | 2011-07-01 |
| Access Restriction | Open |
| Rights Holder | American Society for Investigative Pathology |
| Subject Keyword | Research in Higher Education |
| Content Type | Text |
| Resource Type | Article |
| Subject | Molecular Medicine Pathology and Forensic Medicine |
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