title: DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream "-omic" analyses: possible or not?
abstract: Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.
authors.affiliation: 1Anna Granato, Veterinary and Public Health Institute, viale Università 10, 35020 Legnaro, Padua, Italy. agranato@izsvenezie.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Granato
authors.forename: Mery
authors.initials: M
authors.lastname: Giantin
authors.forename: Pietro
authors.initials: P
authors.lastname: Ariani
authors.forename: Antonio
authors.initials: A
authors.lastname: Carminato
authors.forename: Chiara
authors.initials: C
authors.lastname: Baratto
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zorzan
authors.forename: Marta
authors.initials: M
authors.lastname: Vascellari
authors.forename: Elisa
authors.initials: E
authors.lastname: Bozzato
authors.forename: Mauro
authors.initials: M
authors.lastname: Dacasto
authors.forename: Franco
authors.initials: F
authors.lastname: Mutinelli
mesh_terms: D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D004283:Dog Diseases / Q000235:genetics / Q000473:pathology*; D004285:Dogs; D007150:Immunohistochemistry / Q000662:veterinary; D019394:Ki-67 Antigen / Q000235:genetics; D008415:Mastocytosis / Q000235:genetics / Q000473:pathology*; D016612:Paraffin Embedding / Q000379:methods / Q000662:veterinary*; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D012313:RNA / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary*; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: D019394:Ki-67 Antigen; D051028:bcl-2-Associated X Protein; D012313:RNA; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit
keywords: Cancer; dogs; formalin-fixed, paraffin-embedded tissues; nucleic acids; transcriptomics