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Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue
K Specht1, T Richter, U Müller
1Institute of Pathology, GSF-National Research Center for Environment and Health, Neuherberg, Germany. specht@gsf.de
The American Journal of Pathology
|February 13, 2001
Summary
Formalin-fixed, paraffin-embedded tissues can be reliably used for gene expression analysis. This method, combining laser microdissection and quantitative PCR, enables accurate gene expression profiling from archival samples.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for retrospective studies.
- Genomic analysis of FFPE tissues can elucidate disease mechanisms and identify therapeutic targets.
Purpose of the Study:
- To develop and validate a method for quantitative gene expression analysis in FFPE tissues.
- To assess the accuracy and reproducibility of gene expression measurements from archival samples.
Main Methods:
- Laser-assisted microdissection for isolating specific cell populations.
- Optimized RNA extraction from FFPE tissues.
- Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) using TaqMan methodology.
Main Results:
- Accurate and reproducible gene expression level determinations from FFPE tissues.
- Measurements comparable to fresh-frozen tissues, unaffected by fixation or time.
- Reproducible mRNA quantitation achievable with as few as 50 cells.
- Successful application to HER-2/neu analysis in Barrett's esophageal adenocarcinoma, correlating with FISH and IHC.
Conclusions:
- The combination of laser microdissection and RT-qPCR is a powerful tool for gene expression analysis in archival FFPE tissues.
- This approach facilitates the investigation and clinical validation of gene expression changes in patient samples.