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Updated: Aug 23, 2026
Laser Capture Microdissection of Paraformaldehyde-Fixed Mouse Liver Tissue for RNA Analysis
Published on: April 17, 2026
Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time
Hironori Takagi1, Makoto Shibutani, Natsumi Kato
1Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Abstract:
We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.
Insights
Methacarn fixation preserves mRNA integrity for accurate gene expression analysis in paraffin-embedded tissues (PETs). This method offers performance comparable to unfixed tissues for quantitative mRNA analysis of microdissected specimens.
Area of Science:
- Molecular Biology
- Histology
- Biochemistry
Background:
- Methacarn is a known versatile fixative for analyzing proteins, DNA, and RNA in paraffin-embedded tissues (PETs).
- Quantitative mRNA expression analysis is crucial for understanding cellular function and disease mechanisms.
Purpose of the Study:
- To evaluate the suitability of methacarn fixation for quantitative mRNA expression analysis.
- To assess the fidelity and reliability of gene expression data from microdissected methacarn-fixed PET specimens.
Main Methods:
- Real-time RT-PCR technique was employed for quantitative mRNA expression analysis.
- Methacarn-fixed paraffin-embedded rat liver sections were compared with unfixed frozen tissue.
- Analysis included dose-dependent induction of cytochrome P450 2B1 and effects of hematoxylin staining.
Main Results:
- High fidelity of mRNA expression was observed in methacarn-fixed PET sections, comparable to unfixed frozen tissues, even after hematoxylin staining.
- RNA yield from methacarn-fixed PET sections was equivalent to unfixed cryosections and unaffected by hematoxylin staining.
- Strong correlations (r > 0.98) were found between gene expression levels and RNA input (1-1000 pg), with similar regression curves to unfixed cryosections.
Conclusions:
- Methacarn fixation demonstrates high performance for quantitative mRNA expression analysis in microdissected PET specimens.
- The fixation method yields results closely resembling those from unfixed tissues, making it suitable for sensitive molecular analyses.
- Accurate mRNA quantification requires optimization of cell numbers, with approximately 200 cells recommended for 10-microm rat liver sections.
