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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.
Abstract:
We have found methacarn, a non-crosslinking protein-precipitating fixative, to be useful for the analysis of DNA from microdissected specimens of wax-embedded tissue. In this chapter, we present the procedure regarding genomic DNA analysis in methacarn-fixed wax-embedded microdissected rat tissue. Using nested polymerase chain reaction (PCR), and a rapid extraction procedure, fragments of DNA up to 2.8 kb in size can be amplified from a 1 x 1 mm area of a 10-microm-thick tissue section. Target fragments of about 500 bp can be amplified from a single cell, but 10-20 cells are necessary for practical detection by nested PCR. Although tissue staining with hematoxylin and eosin inhibits the PCR, amplification of about 500-bp fragments is successful with 150-270 cells by single-step PCR. Immunostaining results in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, fragments of about 180 bp can be amplified with 150-270 cells by single-step PCR. These features demonstrate the suitability of methacarn-fixed wax-embedded tissue for practical genomic DNA analysis in terms of tissue handling, extraction efficiency, and satisfactory PCR results.
Insights
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.
