Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.
Author(s): Castiglione F, Degl'Innocenti DR, Taddei A, Garbini F, Buccoliero AM, Raspollini MR, Pepi M, Paglierani M, Asirelli G, Freschi G, Bechi P, Taddei GL
Publication: Appl Immunohistochem Mol Morphol, 2007, Vol. 15, Page 338-42
PubMed ID: 17721281 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to evaluate the effects of differing formalin fixation times (1 to 72 h) on the amplification of GAPDH and COX-2 mRNAs using qRT-PCR.
Summary of Findings:
The authors report successfully extracting RNA from fresh (RNAlater preserved) and formalin-fixed tissues, although RNA extracted from formalin-fixed tissue was more fragmented, 130-200 bp compared to 650 bp in RNAlater preserved controls. They also report successful amplification of mRNA extracted from fresh (RNAlater preserved), formalin-fixed, and formalin-fixed, paraffin-embedded (FFPE) tissues for all fixation times examined; no effects of paraffin embedding were observed on RNA yield or amplification. COX-2 was overexpressed in 30% of tissues formalin -fixed for 1 to 3 h compared to RNAlater preserved controls, and in 75-87% of tissue formalin-fixed for 24 to 72 h. Tissues fixed in formalin for 3 h also exhibited a mean underexpression compared to controls. Thus, the authors conclude that qRT-PCR may be employed to compare mRNA levels between patients only if identical fixation times have been employed.
Biospecimens
Preservative Types
- Formalin
- RNAlater
Diagnoses:
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Electrophoresis RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
RNAlater
Biospecimen Preservation Time in fixative 1 h
3 h
24 h
48 h
72 h