title: Multicentre validation study of nucleic acids extraction from FFPE tissues.
abstract: In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.
authors.affiliation: ACADEM Department-University of Trieste and ICGEB, University of Trieste, Cattinara Hospital, 34149 Trieste, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Bonin
authors.forename: Falk
authors.initials: F
authors.lastname: Hlubek
authors.forename: Jean
authors.initials: J
authors.lastname: Benhattar
authors.forename: Carsten
authors.initials: C
authors.lastname: Denkert
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Fernandez
authors.forename: Gerald
authors.initials: G
authors.lastname: Höfler
authors.forename: Hannelore
authors.initials: H
authors.lastname: Kothmaier
authors.forename: Bozo
authors.initials: B
authors.lastname: Kruslin
authors.forename: Chiara Maria
authors.initials: CM
authors.lastname: Mazzanti
authors.forename: Aurel
authors.initials: A
authors.lastname: Perren
authors.forename: Helmuth
authors.initials: H
authors.lastname: Popper
authors.forename: Aldo
authors.initials: A
authors.lastname: Scarpa
authors.forename: Paula
authors.initials: P
authors.lastname: Soares
authors.forename: Giorgio
authors.initials: G
authors.lastname: Stanta
authors.forename: Patricia J T A
authors.initials: PJ
authors.lastname: Groenen
mesh_terms: D005557:Formaldehyde; D006801:Humans; D009696:Nucleic Acids / Q000032:analysis / Q000302:isolation & purification*; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
chemical_list: D009696:Nucleic Acids; D005557:Formaldehyde