title: Assessment of a fully automated high-throughput DNA extraction method from formalin-fixed, paraffin-embedded tissue for KRAS, and BRAF somatic mutation analysis.
abstract: Preoperative biopsies or imbedded cytological cells will become more and more a primary source of tissue for molecular diagnostic analyses as a result of novel neo-adjuvant treatment regimens for several cancer types. Furthermore there is a growing need to examine metastatic cancer tissue. Hence, nucleic acids need to be reliably isolated and analyzed from small amounts of formalin-fixed and paraffin-embedded (FFPE) tissue. The limited numbers of (tumor) cells in these samples make high quality and sensitive DNA isolation challenging. Also demands for faster turnaround times are growing. Therefore, we evaluated a fully automated DNA/RNA isolation system and compared this with a manual, classical routine molecular pathology method. We compared the quality of the isolates from both tissue cores and micro-dissection for detection of hotspot mutations in KRAS, BRAF applying hydrolysis probe assays. In addition we determined whether the automated method decreases the hands-on-time and turnaround times in routine molecular pathology workflow. In conclusion, the automated method delivers high quality DNA from both small FFPE tissue cores and micro-dissected tissue material. In comparison to classical methods, less than 50% of starting tissue was sufficient as input for micro-dissection. Turnaround times decreased significantly and 50% less hands-on time was needed.
authors: Department of Pathology, Leiden University Medical Center, P.O.box 9600, L1-Q, Albinusdreef 2, 2300 RC Leiden, The Netherlands. r.van_eijk@lumc.nl
authors: Ronald
authors: R
authors: van Eijk
authors: Lisa
authors: L
authors: Stevens
authors: Hans
authors: H
authors: Morreau
authors: Tom
authors: T
authors: van Wezel
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D004247:DNA / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde; D011905:Genes, ras; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012313:RNA / Q000302:isolation & purification; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D012313:RNA; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins