title: Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.
abstract: OBJECTIVE
  Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods.
  
  
  METHODS
  Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared.
  
  
  RESULTS
  Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance.
  
  
  CONCLUSIONS
  FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.
authors.affiliation: From the MRC-Holland BV, Amsterdam, the Netherlands.
authors.forename: Lilit
authors.initials: L
authors.lastname: Atanesyan
authors.affiliation: From the MRC-Holland BV, Amsterdam, the Netherlands.
authors.forename: Maryvonne J
authors.initials: MJ
authors.lastname: Steenkamer
authors.affiliation: Symbiant BV, Alkmaar, the Netherlands.
authors.forename: Anja
authors.initials: A
authors.lastname: Horstman
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Cathy B
authors.initials: CB
authors.lastname: Moelans
authors.affiliation: From the MRC-Holland BV, Amsterdam, the Netherlands.
authors.forename: Jan P
authors.initials: JP
authors.lastname: Schouten
authors.affiliation: From the MRC-Holland BV, Amsterdam, the Netherlands.
authors.forename: Suvi P
authors.initials: SP
authors.lastname: Savola
mesh_terms: D004247:DNA / Q000032:analysis*; D005557:Formaldehyde; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016612:Paraffin Embedding; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D005557:Formaldehyde; D004247:DNA
keywords: Copy number; DNA extraction; FFPE; Formalin fixation; MLPA; Multiplex ligation-dependent probe amplification