P-108: An Efficient Method for DNA Extraction and Purification from Formalin-Fixed and Paraffin-Embedded Autopsied Brain Specimens
Objective: Archival tissue sections are invaluable resources for retrospective molecular genetics and forensic studies. However, extraction of high quality DNA from the autopsy tissues is still a problematic issue due to marked autolysis and high concentrations of proteins and lipids especially in brain specimens. In the present study, we describe a method providing access to polymerase chain reaction (PCR)-amplifiable DNA from formalin-ﬁxed and parafﬁn-embedded (FFPE) autopsied brain specimens. Materials and Methods: Twenty FFPE tissue blocks from brain autopsy tissue were collected between December 2009 and May 2010. Four different DNA extraction methods were evaluated for their ability to obtain an adequate quality and quantity of DNA from tissue specimens. These include methods of heating and non-heating the samples prior to extraction in order to deparaffinization of sections followed by enzymatic digestion. Using microwave-based heating protocol, phenol-chloroform DNA extraction method, commercial CinnaPureTM DNA kit and a non-heating protocol with different concentrations of Proteinase K digestion enzyme coupled with silica-based spin column CinnaPureTM DNA kit, total DNA was extracted and quantified employing spectrophotometer. The quality of extracted DNA was assessed by PCR amplifying a human β-globin housekeeping gene fragment. Results: The best DNA extraction method consisted of deparaffinization by xylene, protein digestion with 25µl of 20 mg/ml Proteinase K at 52°C for 20 hours followed by adding another 10 µl of 20 mg/ml Proteinase K plus 10 µl of ributinase and 100 µl of prelysis solution supplied by CinnaPureTM DNA kit manufacturer incubating at 52°C for an additional 20 hours until the tissue was completely solubilized. Following steps of DNA extraction were performed according to the manufacturer’s protocol. Using this method provided maximum amounts of extracted DNA relating to quality, quantity and number of PCR-amplifiable β-globin DNA fragment cases in comparing with other methods practiced in our assessments. Conclusion: Our study using different protocols for DNA extraction from brain autopsy FFPE specimens indicated that the combination of Proteinase K digestion method and silica-based spin column puriﬁcation protocol results in a higher yield of DNA compared with the three other ordinary methods. However, the protocol presented here, should be validated for various autopsied specimens involving FFPE material before it is considered applicable for molecular studies.