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An efficient and rapid method for DNA extraction from formalin fixed and paraffin embedded tissues suitable for analysis by polymerase chain reaction (PCR). E. Gallo, G. Di Marco, M. Stella, G. Costanzo, M.R. Rizzuto, F. Raiata and A. G. Rizzo Unità Operativa di Anatomia Patologica, Azienda Ospedaliera “V. Cervello”, Palermo Background: • Formalin-fixed paraffin-embedded tissues are a valuable source of DNA for molecular studies due to the availability of large tissue archives in almost all hospital pathology departments. • Reported in the literature there are several methods for DNA extraction from paraffin embedded tissue and most of described methods report an amplification success rate of 60-80%. OBJECTIVE : A wide variety of methods exist for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections and the technical procedures for DNA extraction include many steps such as deparaffinization in xylene and enzymatic digestion (or other chemical treatment coupled with enzyme digestion). In a prospective study we compared two methods to identify an optimal and rapid protocol for DNA extraction from paraffin-embedded tissue samples. MATERIAL AND METHODS: • Tissue samples: Nuclear DNA was extracted from forthy-five cases of surgically resected colorectal adenocarcinomas which had ben fixed in buffered formalin and embedded in paraffin. The sections (10-30mm thick) were cut with standard microtome from every paraffin block and transferred into a 1,5 ml microtube. DNA extraction : DNA extraction was performed using two different methods: a) a commercial kit (Dna extraction kit, BioDiagene) with a simple and rapid protocol b) a conventional method. DNA extraction kit (BioDiagene) • DNA was extracted from 10mm sections of paraffin embedded tissues. • It is a rapid method consisting of xylene/ethanol dewaxing, followed by a kit base extraction without proteinase k digestion. DNA extraction by conventional method • DNA was extracted from 10-30mm sections of paraffin embedded tissues. • The conventional method consisted of xylene/ethanol dewaxing followed by overnight proteinase k digestion in lysis buffer. The sample was heated at 95°C for 5 minutes to inactivate the proteinase k. Genomic DNA PCR We checked the quality of samples by PCR for the housekeeping gene b-globin (fragment of 268 bp). The PCR condition were as follows: denaturation at 94°C for 5 minute, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and extension at 72°C for 1 minute followed by 5 minutes final extension at 72°C. The PCR amplification products were run on 2% agarose gel and stained with ethidium bromide and visualized under ultraviolet light. Results Amplification of the b-globin gene sequence was successful in 42 of 45 (93%) samples using Dna extracted by commercial kit and in 38 of 45 (84,4%) by conventional methods (tab.1). Tab.1- Polymerase chain reaction (PCR) amplification of DNA (b-globin gene) extracted from formalin fixed and paraffin embedded tissue Analysis by two different methods. Methods Total number of PCR samples amplified success Conventional 45 38 (84,4) BioDiagene kit 45 43 (93%) Conclusion Both methods used to obtain genomic DNA from formalin fixed and paraffin-embedded tissues produced DNA suitable for amplification, but the rapid and non- laborious commercial kit could facilitate the molecular retrospective studies. In fact, in only thirty minutes genomic DNA is extracted and made ready to use for PCR amplification.
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