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
Conclusion of Paper
Studies
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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:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 yr
6 yr
17 yr
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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:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Storage Storage duration 1 yr
6 yr
17 yr
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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:
Analyte Technology Platform RNA Real-time qRT-PCR RNA In situ hybridization Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Technology platform Real-time qRT-PCR
Immunohistochemistry
Fluorescent in situ hybridization