Pathology – Research and Practice 208 (2012) 584–591 Contents lists available at SciVerse ScienceDirect Pathology – Research and Practice jo u r n al hom epa ge: www.elsevier.com/locate/prp Original article DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: The effect of formalin fixation and paraffin embedding Karina Silva Funabashi a , Denise Barcelos a , Iria Visoná a , Marcelo Souza e Silva a , Maria Luiza Almeida Prado Oliveira e Sousa a,b , Marcello Fabiano de Franco a , Edna Sadayo Miazato Iwamura a,∗ a Department of Pathology – Escola Paulista de Medicina, Universidade Federal de São Paulo – EPM/UNIFESP, São Paulo, SP, Brazil b Laboratorio de Biologia e Bioquímica – Instituto de Criminalistica de São Paulo, São Paulo, SP, Brazil a r t i c l e i n f o Article history: Received 9 April 2012 Received in revised form 29 May 2012 Accepted 5 July 2012 Keywords: Molecular pathology Autopsy Formalin-fixed Paraffin-embedded PCR STR a b s t r a c t The use of molecular biology in combination with morphological analysis is increasing because of the treatments by target therapies. However, to improve the methods for obtaining DNA for molecular anal- yses from formalin-fixed, paraffin-embedded (FFPE) tissue is a challenge. The aim of this study was to evaluate the DNA extracted from FFPE tissue blocks (non-tumoral liver, spleen, and brain), obtained from autopsy, 8–24 h post mortem, using three methods of DNA extraction. PCR of the -actin (136pb) and human amelogenin (AMEL 212–218 bp/106–112 bp) genes, as well as short tandem repeat (STR) (100–400 bp fragments), reported in forensic scientific analysis, was performed to evaluate the effective- ness of the methods of DNA extraction. We used 28 archived (1 and 5 years) and 12 recent autopsy cases. The commercial kit showed reproducible and consistent results in the PCR amplification of the -actin and AMEL genes and in analysis by STR used in forensic analysis. This is the first report using non-tumoral samples from FFPE autopsy tissues, comparing the three most common methods of DNA extraction and using the STR previously described in forensics. Our study has clarified the challenges for pathologists in applying the molecular biology approach in combination with methods suited for morphology, which must be improved. The data provided here should be used in other molecular studies in FFPE samples. © 2012 Elsevier GmbH. All rights reserved. Introduction The process of formalin fixation and paraffin embedding (FFPE) is considered a standard method of preservation of biological sam- ples for long periods. This type of sample is important for medical diagnosis [1], retrospective genetic and epidemiological studies [2], studies of rare diseases [3] and use in forensics [4–7] and is an excellent source of DNA [8,9], although its recovery for molecular analysis is still a challenge [10]. The processing of paraffin samples, although standardized, can vary according to each laboratory protocol, the type of fixatives, fixation time, and other reagents [11]. Because the penetration rate of 10% formalin is approximately 1 mm/h, the tissue should not be exposed to the fixative for longer than necessary, as this can cause damage to nucleic acids. Extensive cross-links between proteins ∗ Corresponding author at: Departamento de Patologia – Escola Paulista de Medic- ina, Universidade Federal de São Paulo, Rua Botucatu, 740 edifício Lemos Torres, Vila Clementino – CEP 04023-062, São Paulo, SP, Brazil. Tel.: +55 11 5576 4996/848. E-mail address: eiwamura@gmail.com (E.S.M. Iwamura). in the tissues and DNA fragmentation can be found when fixing is performed for too long, leading to lower quality DNA [12]. There are several protocols for DNA extraction from fresh tissues, blood, and cell culture. However, DNA extraction from paraffin-embedded tissue requires special protocols because the material is often scarce, degraded, and can contain substances that inhibit the molecular procedures [13]. Among the extraction methods used for FFPE samples, the simplest and most afford- able is Salting-Out, which uses the insolubility of long strands of DNA in a specific salt concentration [14]. The phenol–chloroform method, a process of traditional DNA extraction that involves the removal of proteins and other cellular components by the action of phenol dissolved in chloroform [15], can also be used. Finally, the DNA filtering method uses silica, which can adsorb nucleic acids, which are dependent on the salt concentration and pH [16]. Polymerase chain reaction (PCR) is a specific and sensitive molecular technique of relatively fast execution and low cost that allows for the detection of specific DNA fragments. In the litera- ture, it has been reported that the average fragment length of DNA is 300–400 bases in biopsy tissues, but much smaller in FFPE tis- sues from autopsy [17]. This limits the use of FFPE samples for 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2012.07.001