Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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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

This paper sought to determine whether the duration of paraffin block storage affects the integrity of RNA extracted from formalin-fixed paraffin-embedded specimens (FFPE), as well as to evaluate expression profiles among specimens with regard to different preservation methods (frozen versus formalin fixation), cancer status (cancerous versus normal adjacent), and biospecimen locations via the cDNA-mediated annealing, selection, extension, and ligation (DASL) assay and microarray analysis.

Conclusion of Paper

Storage of formalin fixed specimens embedded in paraffin blocks adversely affected RNA integrity in a time dependent manner. Reproducible expression profiles were obtained via DASL and microarray analysis using as little as 50 ng of total RNA extracted from FFPE specimens. While the number of genes identifiable among fresh frozen and FFPE specimens were comparable, the number of genes differentially expressed among cancerous and normal adjacent specimens varied among preservation methods (28 vs. 16%, respectively). However, cluster analysis revealed that analysis of a subset of genes successful segregated specimens based upon cancer status and biospecimen location independent of preservation method.

Studies

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

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

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

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

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