RNA Quantity and Quality from Paraffin Blocks: A Comparison of Fixation, Processing and Nucleic Acid Extraction Techniques.
Gulisa Turashvili, Winnie Yang, Stephen Yip, Melinda Carrier, Nadia Gale, Ying Ng, Katie Chow, Lynda Bell, Margaret Luk, Steve Kalloger, Blake Gilks, Samuel Aparicio, David Huntsman. BC Cancer Agency, Provincial Health Services Authority, Vancouver, BC, Canada; Vancouver General Hospital and University of British Columbia, BC, Canada
Background: RNA extracted from paraffin blocks can be used for clinical molecular diagnostic assays, including RT-PCR, quantitative PCR, cDNA library construction and most recently whole transcriptome shotgun sequencing. Although the capacity to extract high quality RNA from paraffin blocks is crucial, there is little information available to guide laboratories in their selection of tissue fixation, processing and RNA extraction techniques.
Design: Nine human tissue samples (three each of colon, liver and muscle) were subjected to the following fixation and processing conditions: (1) flash freezing; (2) neutral buffered formalin (NBF) fixation <24 hours (NBF24); (3) NBF fixation 7 days (NBF7); (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). Total RNA was extracted using phenol-chloroform manual extraction, RecoverAll (Ambion, USA), Waxfree RNA (Trimgen, USA), and RNeasy FFPE Kit (Qiagen, Canada). RNA was quantified using a Nanodrop spectrophotometer, and one-step RT-PCR was used for the amplification of two ACTB fragments (621 bp and 816 bp).
Results: Manual extraction and WaxFree RNA kit yielded higher amounts of RNA when compared with RNeasy and RecoverAll kits, independent of the type of tissue, fixation or processing. For frozen tissues, there was no difference between manual extraction and WaxFree RNA kit for 621 bp amplicon but manual extraction performed better for 816 bp amplicon (p<0.001). In MF-fixed tissues, both amplicons were successfully amplified using all extraction methods except for WaxFree RNA kit. For NBF24 tissues, 621 bp amplicon was amplified using all kits but manual extraction performed better for the 816 bp amplicon (p<0.001). For NBF7-fixed tissues, manual extraction and WaxFree RNA kit were superior to RNeasy and RecoverAll kits (p<0.01). None of the extraction kits succeeded in amplifying the 816 bp amplicon in a majority of NBF7 samples.
Conclusions: The molecular fixative, regardless of the duration of fixation, and the rapid processing system used in this study were able to preserve RNA in paraffin blocks with successful RT-PCR for as long as 816 bp amplicons, making these techniques suitable for use in downstream molecular diagnostic assays.
Monday, February 28, 2011 1:00 PM
Poster Session II # 268, Monday Afternoon