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Methacarn fixation for genomic DNA analysis in microdissected cells
Makoto Shibutani1, Chikako Uneyama
1Division of Pathology, National Institute of Health Sciences, Tokyo, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|July 21, 2005
Summary
Methacarn fixative enables genomic DNA analysis from microdissected, wax-embedded rat tissues. This method allows DNA amplification from small tissue samples, even after common staining procedures.
Area of Science:
- Molecular Biology
- Genomics
- Histology
Background:
- Wax-embedded tissues are common in research but pose challenges for DNA analysis.
- Traditional fixatives can degrade DNA or hinder downstream applications.
- Methacarn offers a non-crosslinking alternative for tissue fixation.
Purpose of the Study:
- To present a procedure for genomic DNA analysis in methacarn-fixed, wax-embedded microdissected rat tissue.
- To evaluate the efficiency of DNA extraction and polymerase chain reaction (PCR) amplification from such samples.
- To assess the impact of common histological staining methods on DNA analysis.
Main Methods:
- Microdissection of methacarn-fixed, wax-embedded rat tissue sections (10-microm thick).
- Rapid DNA extraction procedure.
- Nested polymerase chain reaction (PCR) for DNA amplification.
- Analysis of DNA fragments up to 2.8 kb.
Main Results:
- DNA fragments up to 2.8 kb can be amplified from 1 mm² of tissue.
- Nested PCR can amplify target fragments (approx. 500 bp) from as few as 10-20 cells.
- Single-step PCR successfully amplifies ~500 bp fragments from 150-270 cells, even after hematoxylin and eosin staining.
- DNA fragments (~180 bp) are amplifiable via single-step PCR from 150-270 cells post-immunostaining.
Conclusions:
- Methacarn is suitable for genomic DNA analysis of microdissected, wax-embedded tissues.
- The method demonstrates good tissue handling, extraction efficiency, and PCR results.
- This technique supports practical genomic DNA analysis from challenging tissue samples.