NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Author(s): Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M

Publication: PLoS One, 2007, Vol. 2, Page e537

PubMed ID: 17579711 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to compare methods for the isolation of DNA and RNA from formalin-fixed and Bouin's-fixed paraffin-embedded (FFPE and BFPE) specimens.

Conclusion of Paper

While there was no effect of deparaffinization, lithium carbonate washing or critical point drying, protocols that included incubation of the specimens at 98 degrees C for 15 minutes prior to proteinase k digestion, proteinase K digestion at 65 degrees C for 48 h, extraction of the DNA and RNA with QIAamp DNA kit and DNA repair with Taq led to the highest amplifiable nuclear DNA (nuDNA), mitochondrial DNA (mtDNA) and RNA and/or improved amplification of proviral DNA or viral RNA when compared to other methods. Hot alkali treatment (incubation in a pH 11.2 buffer at 120 degrees C) instead of proteinase K digestion increased amplifiable DNA, but led to RNA degradation. The addition of glycine to proteinase k digestion buffer led to a decrease in amplifiable DNA.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of isolation method on DNA and RNA yield, amplifiable yield of nuDNA, mtDNA, RNA, proviral DNA and viral RNA, DNA size ratio, and SNP detection by multiplex PCR microsequencing from FFPE and BFPE Kaposi's sarcoma, lymphoma, lung, and spleen specimens. The effects of deparaffinization, incubation at 98 degrees C prior to proteinase k digestion, lithium carbonate washing, proteinase k digestion buffer composition, proteinase k digestion temperature and duration, extraction method, taq based DNA repair, hot alkali treatment, and critical point drying were examined.

    Summary of Findings:

    There was no effect of deparaffinization or critical point drying protocols on any of the parameters tested. Incubation of specimens at 98 degrees C for 15 minutes prior to proteinase K digestion increased the amplification of HIV-1 by RT-PCR, the amount of amplifiable nuDNA, and decreased multiplex PCR with minisequencing success without altering DNA or RNA yield, quantity of amplifiable mtDNA or RNA, or DNA amplicon size ratio. Addition of glycine to the proteinase k digestion buffer significantly reduced the amount of amplifiable nuDNA and mtDNA. Increasing digestion time from 24 h to 48 h led to an increase in amplifiable nuDNA and RNA. The authors state that digestion beyond 48 h increased amplifiable yields in some specimens, but the effect was variable and some specimens showed decreased amplifiable yield with extended digestion. Digestion at 65 degrees C instead of 55 degrees C increased yield of amplifiable RNA and amplification of proviral DNA, but further increasing the incubation temperature to 75 or 85 degrees C led to DNA and RNA degradation. Incubation at 120 degrees C in buffer with a pH of 11.2 (or in 0.1 M NaOH) increased the amount of amplifiable DNA but decreased total DNA yield and increased RNA degradation compared to specimens incubated for 48 h with proteinase K at 65 degrees C. Extraction of DNA and RNA with QIAamp DNA extraction kit led to increased yield of amplifiable RNA and nuDNA, but did not affect the amplifiable DNA size ratio. Repairing the DNA by incubation with Taq did not affect the amplifiable DNA size ratio, but increased the quantity of amplifiable nuDNA. In the small number of specimens examined, there was no significant effect of lithium carbonate washing on BFPE specimens, but there was a nonsignificant decrease in DNA and RNA yield (47-58%, and 36-60% respectively).

    Biospecimens
    Preservative Types
    • Formalin
    • Other Preservative
    Diagnoses:
    • Neoplastic - Lymphoma
    • Neoplastic - Sarcoma
    • AIDS/HIV-related
    Platform:
    AnalyteTechnology Platform
    DNA PCR
    DNA Real-time qPCR
    DNA SNP assay
    RNA Real-time qRT-PCR
    RNA RT-PCR
    RNA Spectrophotometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    Formalin (unbuffered)
    Bouin's fixative
    Analyte Extraction and Purification Analyte isolation method Inclusion of dehydration and critical point drying (several protocols)
    QIAamp extraction
    Phenol-chloroform extraction
    Inclusion of DNA repair with taq polymerase
    Analyte Extraction and Purification Washing LiCO3 wash
    None
    Analyte Extraction and Purification Protein digestion 55 degrees C
    65 degrees C
    75 degrees C
    85 degrees C
    Buffer containing 25 mM glycine
    Standard buffer
    Hot alkali treatment (varied pH and temperature)
    None (only hot alkali treatment),
    1 h
    6 h
    12 h
    24 h
    48 h
    72 h
    PCR Specific Targeted nucleic acid Amelogenin
    HIV gag gene
    PCR Specific Length of gene fragment 106/112
    212/218
    Real-time qRT-PCR Specific Targeted nucleic acid Mitochondrial DNA hypervariable region
    Amelogenin
    RT-PCR Specific Targeted nucleic acid Beta 2 microglobulin
    HIV-1
    Analyte Extraction and Purification Deparaffinization None
    Xylene
    Pentane

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