title: Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma.
abstract: Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.
authors.affiliation: Laboratory of Bacteriology, Gastrocenter, State University of Campinas, Campinas, São Paulo, Brazil. Electronic address: elizabeth.goncalves@gc.unicamp.br.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rabelo-Gonçalves
authors.affiliation: Laboratory of Bacteriology, Gastrocenter, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Bruna
authors.initials: B
authors.lastname: Roesler
authors.affiliation: Laboratory of Bacteriology, Gastrocenter, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Guardia
authors.affiliation: Laboratory of Bacteriology, Gastrocenter, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Arlete
authors.initials: A
authors.lastname: Milan
authors.affiliation: Laboratory of Bacteriology, Gastrocenter, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Natalicia
authors.initials: N
authors.lastname: Hara
authors.affiliation: Department of Anatomic Pathology, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Cecília
authors.initials: C
authors.lastname: Escanhoela
authors.affiliation: Gastrocenter, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Jazon
authors.initials: J
authors.lastname: Almeida
authors.affiliation: Gastrocenter, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: Ilka
authors.initials: I
authors.lastname: Boin
authors.affiliation: Gastrocenter, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil.
authors.forename: José Murilo
authors.initials: JM
authors.lastname: Zeitune
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D016481:Helicobacter Infections / Q000175:diagnosis / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012189:Retrospective Studies; D021521:Ribotyping; D013048:Specimen Handling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D005404:Fixatives; D012336:RNA, Ribosomal, 16S; D005557:Formaldehyde
keywords: 16S Ribosomal RNA; DNA; Helicobacter pylori; Liver; Paraffin embedded