DNA Quantity and Quality from Paraffin Blocks: A Comparison of Fixation, Processing and Nucleic Acid Extraction Techniques
G Turashvili, M Carrier, N Gale, Y Ng, K Chow, L Bell, M Luk, S Kalloger, B Gilks, S Aparicio, D Huntsman. BC Cancer Agency, Provincial Health Services Authority, Vancouver, BC, Canada; Vancouver General Hospital, University of British Columbia, Canada, BC, Canada
Background: DNA extracted from paraffin blocks can be used for mutation detection, copy number analysis and most recently whole genome or exome sequencing using Next Generation Sequencing. Although the capacity to extract high quality DNA from paraffin blocks is of more importance now than ever, there is little information available to guide laboratories in their selection of tissue fixation, processing and DNA extraction techniques.
Design: Nine samples (three each of colon, liver and muscle) were subjected to the following fixation and processing conditions: 1. frozen; 2. neutral buffered formalin (NBF) fixation <24 hours; 3. NBF fixation 7 days; 4. molecular fixative (MF) <24 hours; 5. MF 7 days. NBF-fixed samples were processed by standard processing (Tissue-Tek VIP5 processor, Somagen, Canada), and MF-fixed samples by rapid processing (Tissue-Tek Xpress, Somagen). DNA was extracted using phenol-chloroform manual extraction, RecoverAll (Ambion, USA), Waxfree DNA (Trimgen, USA), and QIAamp DNA FFPE Tissue Kit (Qiagen, Canada). DNA quantity and quality were assessed using Nanodrop, and PCR was attempted for amplicons of five lengths (268-1300 bp).
Results: Manual extraction yielded the highest amount of DNA followed by Waxfree DNA, QIAamp and finally RecoverAll kit, independent of the type of tissue, fixation or processing. For all five amplicons used, there were no significant differences between MF-fixed and frozen tissues, and between manual and Waxfree DNA kits on PCR. High molecular weight (1300 bp) DNA was preserved in MF-fixed tissues stored for <24 hours or 7 days, with no difference among the four kits (p>0.05). For NBF-fixed tissues, all kits performed similarly except for RecoverAll kit and manual extraction which amplified 536-989 bp and 536 bp fragments, respectively, in fewer samples than other kits (p<0.02). Short DNA fragments (268 bp) were successfully amplified in all tissue types using all kits.
Conclusions: The molecular fixative regardless of the duration of fixation, and the rapid processing system used in this study were able to preserve large DNA fragments in paraffin blocks, making these techniques suitable for use in molecular assays, particularly those involving sequencing technologies.
Monday, March 22, 2010 1:00 PM
Poster Session II # 244, Monday Afternoon