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Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens
Chikako Uneyama1, Makoto Shibutani, Naoya Masutomi
1Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
Abstract:
We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.
Insights
Methacarn fixative enables versatile genomic DNA analysis from microdissected paraffin-embedded tissues (PET). This method allows extensive DNA amplification, even from single cells, outperforming traditional formalin fixation.
Area of Science:
- Molecular Biology
- Genomics
- Histology
Background:
- Methacarn is a versatile fixative for RNA and protein analysis in microdissected paraffin-embedded tissue (PET).
- Its efficacy for genomic DNA analysis in such specimens requires further investigation.
Purpose of the Study:
- To evaluate methacarn's performance for genomic DNA analysis in microdissected rat tissues.
- To compare methacarn with formalin for DNA analysis in PET specimens.
Main Methods:
- DNA extraction from 1 mm³ microdissected rat cerebral cortex sections (10 µm thick).
- Nested PCR and single-step PCR for DNA amplification.
- Analysis of DNA amplification from single cells and small cell populations (10-270 cells).
- Assessment of DNA quality and yield after various staining procedures (cresyl violet, hematoxylin and eosin, immunostaining).
Main Results:
- Extensive DNA portions (up to 2.8 kb) were amplified using nested PCR.
- A 522-bp fragment was amplified from single Purkinje cells (20% success rate).
- Minimal cell requirement was 10-20 cells for hippocampal neurons.
- Hematoxylin and eosin staining allowed 522-bp amplification from 150-270 cells.
- Immunostaining reduced DNA yield but allowed 184-bp fragment amplification from 150-270 cells.
Conclusions:
- Methacarn demonstrates superior performance for genomic DNA analysis in microdissected PET specimens compared to formalin.
- Methacarn-fixed tissues support robust DNA amplification across various staining protocols, including single-cell analysis.