title: Optimizing gDNA extraction from fresh frozen meningioma tissue for downstream genetic analysis.
abstract: OBJECTIVE
  Meningioma is the most common brain tumor. Genetic mutations in meningioma that include deletion of the neurofibromatosis type 2 gene, (NF2), offer diagnostic information on tumor behavior, recurrence and potential response to treatment. Obtaining high-grade genetic material is critical for accurate, sensitive and robust molecular testing. Currently, no standardized procedure exists for extracting gDNA from meningioma, and this problem was addressed in this report.
  
  
  METHODS
  This study compared the yield and quality of extracted gDNA from patient meningioma specimens using an optimized phenol chloroform method and two commercial silica column-based extractions kits and tested respective performances as template in qPCR tests and multiplex ligation-dependent probe amplification (MLPA) NF2 screening.
  
  
  RESULTS
  Mean gDNA yields were comparable for each method tested; however, phenol chloroform extraction outperformed column-based kits in all other quality assurance metrics examined. Phenol chloroform extracted gDNA was highly pure, and of a higher fragment size species when compared to column prepared gDNA. qPCR of GAPDH, B2MG, and RPL37A housekeeping genes demonstrated variance in cycle thresholds between patient samples was much lower in the phenol chloroform group. Similarly, primer efficiencies were significantly improved in this sample group which translated to a broader qPCR linear dynamic range and much improved qPCR performance at low concentrations of template. MLPA screening identified NF2 gene deletions in 6 of 12 meningioma samples. Inconsistencies in copy number data for NF2 and reference regions of the genome were observed between gDNA sample extraction groups that included both false negative and positive errors in silica column derived gDNA samples.
  
  
  CONCLUSIONS
  This study outlines a highly robust phenol chloroform extraction method for obtaining high-quality gDNA from frozen meningioma tissue and highlights the significance of performing adequate quality assurance when using gDNA for downstream genetic analysis. Most importantly, we demonstrate using gDNA extracted with silica column based kits can lead to diagnostic errors when screening NF2 deletions in meningiomas with MLPA.
authors.affiliation: Project neuroArm, Department of Clinical Neuroscience, and the Hotchkiss Brain Institute, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada. Electronic address: dtprocto@ucalgary.ca.
authors.forename: D T
authors.initials: DT
authors.lastname: Proctor
authors.affiliation: Project neuroArm, Department of Clinical Neuroscience, and the Hotchkiss Brain Institute, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada.
authors.forename: E H
authors.initials: EH
authors.lastname: Yoo
authors.affiliation: Project neuroArm, Department of Clinical Neuroscience, and the Hotchkiss Brain Institute, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Vujadinovic
authors.affiliation: Project neuroArm, Department of Clinical Neuroscience, and the Hotchkiss Brain Institute, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Lama
authors.affiliation: Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada.
authors.forename: G
authors.initials: G
authors.lastname: van Marle
authors.affiliation: Project neuroArm, Department of Clinical Neuroscience, and the Hotchkiss Brain Institute, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada.
authors.forename: G R
authors.initials: GR
authors.lastname: Sutherland
mesh_terms: D000328:Adult; D000368:Aged; D015925:Cryopreservation; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D015894:Genome, Human; D006801:Humans; D008297:Male; D008577:Meningeal Neoplasms / Q000235:genetics*; D008579:Meningioma / Q000235:genetics*; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D058996:Quality Improvement; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004273:DNA, Neoplasm
keywords: Brain tumor; DNA extraction; MLPA; Meningioma; NF2; qPCR