Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays.
Author(s): Bibikova M, Talantov D, Chudin E, Yeakley JM, Chen J, Doucet D, Wickham E, Atkins D, Barker D, Chee M, Wang Y, Fan JB
Publication: Am J Pathol, 2004, Vol. 165, Page 1799-807
PubMed ID: 15509548 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine if the duration of paraffin block storage (1-11 years) influences the integrity of RNA extracted from formalin fixed cancerous and normal breast and colon specimens.
Summary of Findings:
RNA integrity, as determined by real-time quantitative RT-PCR (qRT-PCR) amplification of 90 bp and 155 bp fragments of the RPL 13A genes, was adversely affected by the duration of paraffin block storage. Paraffin blocks (containing formalin fixed specimens) stored for 11 years differed from those stored for 1 year by as many as 6 PCR cycles.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 yr
2 yr
9-11 yr
Real-time qRT-PCR Specific Length of gene fragment 90 bp
155 bp
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Study Purpose
The purpose of this study was to determine if the quantity of RNA template impacts DASL performance and reproducibility for analysis of FFPE or fresh-frozen colon and breast tissues.
Summary of Findings:
Reproducible expression profiles were obtained with as little as 50 ng of total RNA as template for the DASL assay from FFPE specimens. Approximately 90% of the genes detected in fresh frozen specimens were also present in FFPE specimens. Although the number of genes differentially expressed between cancerous and normal adjacent case-matched specimens was more robust in fresh frozen specimens versus FFPE specimens (28 vs. 16%, respectively), there was a high degree of overlap in terms of gene identity among frozen and FFPE specimens.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA DASL RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) DASL Specific Template/input amount 50 ng
100 ng
250 ng
500 ng
1000 ng
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
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Study Purpose
The purpose of this study was to compare real-time qRT-PCR threshold cycle number and DASL array intensity for cancer-specific genes in fresh-frozen and FFPE breast and colon tissues.
Summary of Findings:
Of the tissue-specific cancer markers evaluated, real-time qRT-PCR data (expressed as threshold cycle number) correlated well with the intensity of the DASL microarray analysis (R^2=0.88), while correlations were poor for FFPE specimens (R^2=0.41). The authors hypothesize that the poor degree of assay concordance for FFPE specimens is due to sensitivity and reproducibility limitations associated with real-time qRT-PCR.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA DASL RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
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Study Purpose
The purpose of this study was to conduct cluster analysis of expression profiles generated for a subset of genes among case-matched cancerous and normal adjacent specimens preserved by freezing or FFPE.
Summary of Findings:
Analysis of expression profiles via a clustering method revealed all but one specimen segregated based upon diagnosis/ cancer status irrespective of preservation method. A second sample set successfully segregated by both diagnosis/ cancer status and tissue type (colon vs. breast) using FFPE specimens with 100% accuracy. A list of differentially expressed genes is noted in the publication.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA DASL RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Biospecimen Acquisition Biospecimen location Colon
Breast