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A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin fixed, paraffin wax
H E Alcock1, T J Stephenson, J A Royds
1Institute for Cancer Studies, Sheffield University Medical School, UK.
Molecular Pathology : MP
|January 6, 2000
Summary
This study introduces a novel method for genetic analysis of microdissected cells. The technique allows for successful DNA extraction and various polymerase chain reaction (PCR) based analyses on specific cell populations.
Area of Science:
- Molecular Biology
- Genetics
- Histopathology
Background:
- Archival tissue samples are crucial for retrospective studies.
- Immunohistochemistry (IHC) is vital for characterizing cell types in tissues.
- Limited methods exist for performing genetic analysis on IHC-characterized cells.
Purpose of the Study:
- To develop and validate a method for genetic analysis of microdissected cells previously characterized by IHC.
- To enable detailed genetic profiling of specific cell populations within archival tissue specimens.
Main Methods:
- Microdissection of formalin-fixed, paraffin-embedded (FFPE) tissue sections.
- Simple microwave-assisted DNA extraction from microdissected samples.
- Application of various polymerase chain reaction (PCR)-based techniques, including microsatellite analysis and gene sequencing.
- Comparative genomic hybridization (CGH) probe generation.
Main Results:
- Successful DNA extraction and subsequent PCR amplification from microdissected cells.
- Feasibility of microsatellite repeat polymorphism analysis (NM23-H1 locus) and p53 gene sequencing (exons 5, 7, 8).
- Generation of suitable probes for CGH from all microdissected samples.
Conclusions:
- This technique integrates immunohistochemistry with advanced genetic analyses.
- It allows for precise genetic characterization of specific cell subsets identified by IHC.
- This approach significantly enhances the utility of archival FFPE tissues for molecular research.