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Research in Veterinary Science 86 (2009) 421–426

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Research in Veterinary Science
journal homepage: www.elsevier.com/locate/rvsc

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues
Sara Santos a, Daniela Sá a, Estela Bastos a, Henrique Guedes-Pinto a, Ivo Gut b, Fátima Gärtner c,d, Raquel Chaves a,*
aInstitute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (CGB–UTAD/IBB), Quinta de Prados, Apdo 1013, 5001-801 Vila Real, Portugal b CEA/IG - Centre National de Génotypage, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France c Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), University of Porto, Largo Prof. AbelSalazar, 2, 4099-003 Porto, Portugal d Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal

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Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present anadvanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to pre-fixationand fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182 bp. Ó 2008 Elsevier Ltd. All rights reserved.Article history: Accepted 12 August 2008

Keywords: DNA extraction Formalin-fixed paraffin-embedded tissues Cat mammary tumours PCR amplification Sequencing

1. Introduction With growing interest in the genetic basis of diseases the amount of genomic DNA available from biological samples may limit the practicality of genomic analysis (Aviel-Ronen et al., 2006; Gilbert et al., 2007b).Formalin-fixed paraffin-embedded tissues (FFPET) have been used for decades and represent the largest source of archival biological material available for genomic studies (Aviel-Ronen et al., 2006). In some cases, like rare diseases, the FFPET represents the ultimate DNA resource available for genetic analysis that otherwise would be unfeasible (Gilbert et al., 2007a). Previous studies using FFPET of cat mammarytumours (CMT) described molecular changes in oncogenes such as Her2/neu (De Maria et al., 2005; Winston et al., 2005; Morris et al., 2008) or tumour suppressor genes such as p53 (Murakami et al., 2000; Nasir et al., 2000; Morris et al., 2008) by immunohistochemical studies. The detection of mutations in the p53 tumour suppressor gene was previously reported in CMT but in fresh tissues (Mayr etal., 1998). In fact, the nuclear DNA extraction and analysis of cat FFPET has only been reported regarding the amplification of a 108 bp single copy fragment from p53 gene as a positive control for gDNA extraction with a commercial protocol (Kidney et al., 2002). An
* Corresponding author. Tel.: +351 259 350841; fax: +351 259 350572. E-mail address: rchaves@utad.pt (R. Chaves). 0034-5288/$ - seefront matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.08.007

interesting opportunity for comparative oncology has been recognized in naturally occurring tumours in domestic animals (MacEwen, 1990; Vail and MacEwen, 2000) and, more specifically, CMT may be considered a good model for their human counterpart (MacEwen, 1990; Hansen and Khanna, 2004; Zappulli et al., 2005)....