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

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Author(s): Specht Katja, Richter Thomas, Muller Ulrike, Walch Axel, Werner Martin, Hofler Heinz

Publication: Am J Pathol, 2001, Vol. 158, Page 419

PubMed ID: 11159180 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to validate a microscaled RNA extraction method coupled with fluorogenic TaqMan based real-time quantitative RT-PCR for expression analysis of several cancer relevant genes in formalin-fixed paraffin-embedded (FFPE) tissue. Confounding effects associated with delayed fixative penetration of large specimens was also investigated.

Conclusion of Paper

RNA obtained from microdissected FFPE tissue was sufficient for successfully HER-2/neu expression analysis, as results were verified by both FISH and immunohistochemistry. Further, the rate of fixative penetration as a function of specimen size did not significantly affect gene expression.

Studies

  1. Study Purpose

    The purpose of this study was to determine the optimal microscale RNA extraction method for microdissected FFPE archived tissue, and to confirm that quantities are sufficient for real-timer quantitative RT-PCR analysis.

    Summary of Findings:

    The microscale RNA extraction method that resulted in the greatest RNA yield and the most reliable qRT-PCR results for p21/WAF1/Cip1 included proteinase K digestion at 60 degrees C followed by organic extraction. Sufficient mRNA was extracted from FFPE microdissected tumors for successful real-time qRT-PCR analysis of HER-2/neu gene expression; FISH and immunohistochemistry of full tissue sections confirmed real-time qRT-PCR results.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Protein Immunohistochemistry
    DNA FISH
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Real-time qRT-PCR Specific Targeted nucleic acid p21/WAF/Cip1
    HER-2/neu
    Analyte Extraction and Purification Analyte isolation method Proteinase K digestion at 60 degrees C and organic extraction
    Proteinase K digestion at 42 degrees C
    Acidic guanidinium thiocyanate-phenol chloroform
    Oligo d(T) coupled magnetic beads
    Guanidinium thiocyanate lysis with silica-matrix spin technology
    Biospecimen Aliquots and Components Aliquot size/volume Microdissected cells
    Full FFPE section
  2. Study Purpose

    The purpose of this study was to determine possible effects on gene expression (EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, HPRT, PGK) of delayed fixation as a function of the rate of fixative penetration in large specimens (>7 cm) that were fixed in formalin for 20 h. Liver and uterus specimens were analyzed.

    Summary of Findings:

    Dissected regions from the periphery to the core of the specimen did not differ in expression for any of the following 5 genes analyzed by RT-PCR: EGFR, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    RNA RT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    RT-PCR Specific Targeted nucleic acid EGFR
    HER-2/neu
    FGF-R4
    p21/WAF1/Cip1
    MDM2
    Biospecimen Aliquots and Components Biospecimen heterogeneity Biospecimen core
    Biospecimen periphery

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