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

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

Author(s): Chu Wei-Sing, Liang Qi, Liu Jilan, Wei Min Q, Winters Mary, Liotta Lance, Sandberg Glenn, Gong Maokai

Publication: Lab Invest, 2005, Vol. 85, Page 1416

PubMed ID: 16127423 PubMed Review Paper? No

Purpose of Paper

Validation of a novel nondestructive molecule extraction (NDME) method for fixed or frozen tissue sections that liberates proteins and nucleic acids for immediate molecular analysis while preserving cellular structure of the tissue section.

Conclusion of Paper

Nucleic acid and protein extraction of frozen and formalin fixed paraffin embedded (FFPE) tissue sections using the NDME method yielded high quality DNA, RNA, and protein sufficient for successful PCR, ISH, Western blot, immunohistochemistry, and mass spectrometry analyses. In most cases, NDME treatment enhanced the sensitivity of the technology platform. Further, the cellular structure of the tissue section was not compromised with extraction incubations of 20 min or less. In general, the lower nucleic acid yields observed with FFPE tissue sections could not be remedied by NDME extraction.

Studies

  1. Study Purpose

    To access the yield and size of nucleic acids and proteins extracted from FFPE or frozen tissue section using the NDME method. Tissues examined include brain, breast, heart, pancreas, liver, lung, spleen, lymph node, colon, and prostate.

    Summary of Findings:

    As determined by coomassie blue and silver staining, protein and nucleic acid yields were significantly greater compared to other extraction buffers, and included high molecular weight species. Nucleic acid yield was reduced (75%) in extracts from FFPE tissue sections compared to frozen sections.

    Biospecimens
    Preservative Types
    • Formalin
    • OCT
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Protein 1D/2D gels
    RNA Electrophoresis
    DNA Electrophoresis
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    OCT
    Analyte Extraction and Purification Analyte isolation method NMDE-PE
    FFPE-NE
    0.01 M EDTA
    10 mM citrate
    Tissue-PE LB
  2. Study Purpose

    To determine if cellular structure is compromised after NDME treatment of tissue sections.

    Summary of Findings:

    Cellular structure remained preserved after NDME extraction. Further, hematoxylin and eosin staining was enhanced after NDME treatment, with more vivid staining, reduction of intercellular spaces, and slight swelling of nucleoli.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Morphology H-and-E microscopy
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
  3. Study Purpose

    To assess the effects of NDME treatment on CD5 antigenicity as determined by immunohistochemistry.

    Summary of Findings:

    CD5 immunopositive staining revealed that antigen retrieval was not necessary in NDME treated tissue sections. Both the intensity of the immunohistochemical signal and the protein yield in the extraction buffer increased with the length of extraction, plateauing at 30 min, however, incubations longer than 20 min compromised structural integrity of the tissue section.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Protein Immunohistochemistry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Incubation duration/condition 0 min
    5 min
    10 min
    15 min
    20 min
    30 min
  4. Study Purpose

    To determine if protein size and antigenicity are compromised after NDME.

    Summary of Findings:

    Western blot analysis for representative membrane glycoproteins, HIV capsid, Golgi precursor, and nuclear proteins displayed appropriate bands at the predicted sizes. Futher, use of NDME-samples appears to increase platform sensitivity, with a single tissue section extract able to generate a detectable Western blot signal. Microarray analysis of NDME treated protein extracts revelaed expression of common proteins in the majority of tissues examined, as well as appropriate expression of tissue-specific proteins.

    Biospecimens
    Preservative Types
    • Formalin
    • Ethanol
    • Other Preservative
    • OCT
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Protein Western blot
    Protein Reverse phase protein microarray
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation NDME
    Ethanol
    Formalin (buffered)
    OCT
  5. Study Purpose

    To determine if protein profiles are compromised after NDME.

    Summary of Findings:

    Extracts from frozen and FFPE sections (enriched and desalted) exhibited similar protein profiles, within low and high (10-21K m/z) mass/charge (m/z) ranges.

    Biospecimens
    Preservative Types
    • Formalin
    • OCT
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Protein SELDI-TOF MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method NDME
    Biospecimen Preservation Type of fixation/preservation OCT
    Formalin (buffered)
  6. Study Purpose

    To determine if the integrity of NDME nucleic acids is sufficient for molecular analysis.

    Summary of Findings:

    Successful PCR analysis of DNA and RNA extracted using the NDME method indicated nucleic acid integrity was preserved (amplicon size range: 354-1309 bp). However, mRNA quantity was significantly lower (50%) in FFPE sections compared to frozen tissue sections. In situ hybridization post NDME yielded appropriate signal localization and preservation of cellular detail.

    Biospecimens
    Preservative Types
    • Formalin
    • OCT
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    DNA PCR
    DNA In situ hybridization
    RNA In situ hybridization
    RNA RT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    OCT

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