title: RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods.
abstract: BACKGROUND
  Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required.
  
  
  METHODS
  We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a).
  
  
  RESULTS
  The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h).
  
  
  CONCLUSIONS
  The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.
authors.affiliation: Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Bohmann
authors.forename: Guido
authors.initials: G
authors.lastname: Hennig
authors.forename: Uwe
authors.initials: U
authors.lastname: Rogel
authors.forename: Christopher
authors.initials: C
authors.lastname: Poremba
authors.forename: Berit Maria
authors.initials: BM
authors.lastname: Mueller
authors.forename: Peter
authors.initials: P
authors.lastname: Fritz
authors.forename: Stephan
authors.initials: S
authors.lastname: Stoerkel
authors.forename: Karl-L
authors.initials: KL
authors.lastname: Schaefer
mesh_terms: D001331:Automation; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics; D005557:Formaldehyde; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D016612:Paraffin Embedding; D012313:RNA / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013997:Time Factors; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde; D012313:RNA