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

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

Author(s): Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB

Publication: Am J Pathol, 2004, Vol. 164, Page 35-42

PubMed ID: 14695316 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine if RNA fragmentation associated with formalin fixation and paraffin embedding (FFPE) is (1) correlated to the age of the paraffin block, and (2) precludes accurate expression analysis by real-time quantitative RT-PCR (qRT-PCR).

Conclusion of Paper

The authors conclude that real-time qRT-PCR analysis of archival FFPE specimens generates biologically relevant expression data when expression is normalized to that of a reference gene set, and amplicon length remains constant and short (85 bp or less) for all genes examined.

Studies

  1. Study Purpose

    The purpose of this study was to assess the degree of RNA fragmentation in extracts from FFPE tissue stored for varying lengths of time as paraffin blocks.

    Summary of Findings:

    While RNA fragmentation was observed among all FFPE specimens, those stored as paraffin blocks for 6 years or longer yielded RNA in a lower molecular weight range than those stored for 1 year or less.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Automated electrophoresis/Bioanalyzer
    RNA Fluorometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 1 yr
    6 yr
    17 yr
  2. Study Purpose

    The purpose of this study was to identify analytical parameters that would permit gene expression comparisons among FFPE specimens stored for different durations and frozen specimens.

    Summary of Findings:

    Raw Ct values for 92 targeted genes was positively correlated to the duration of paraffin block storage for FFPE specimens, indicating that the quantity of amplifiable RNA decreased with increasing storage duration. This decrease in expression was attenuated by normalizing expression to that of a reference gene set. A separate experiment using case-matched frozen and FFPE specimens confirmed these results. Amplification was adversely affected by an amplicon length of 90 bp or more, with amplicons of 70 to 85 bp yielding optimal results. Individual analysis of a subset of genes confirmed a predicted overexpression of breast cancer inducible genes.

    Biospecimens
    Preservative Types
    • Formalin
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    Snap frozen
    Storage Storage duration 1 yr
    6 yr
    17 yr
  3. Study Purpose

    The purpose of this study was to validate real-time qRT-PCR results for mRNA expression patterns by examining mRNA and protein expression via fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), respectively.

    Summary of Findings:

    Real-time qRT-PCR results for mRNA expression correlated well with IHC results for protein expression, as well as FISH analysis of mRNA levels with the exception of a few older FFPE specimens. Of note, the length of the FISH probe was not provided.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    RNA In situ hybridization
    Protein Immunohistochemistry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Real-time qRT-PCR Specific Technology platform Real-time qRT-PCR
    Immunohistochemistry
    Fluorescent in situ hybridization

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