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Updated: May 11, 2026
Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages
Published on: April 11, 2025
RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section
Yurika Kimura1, Sachiho Kubo, Hiroko Koda
1Department of Otolaryngology, Tokyo Metropolitan Geriatric Medical Hospital, 35-2, Itabashi, Tokyo 173 0015, Japan. kimura@tmghig.jp
Objective:
Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection.
Methods:
Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone.
Results:
COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases.
Conclusion:
We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases.
Insights
Researchers successfully extracted mRNA from archival human inner ear tissues using laser microdissection and RT-PCR. This method enables molecular analysis of otological diseases in preserved specimens.
Area of Science:
- Otolaryngology
- Molecular Biology
- Genetics
Background:
- Archival human inner ear specimens present challenges for molecular analysis due to anatomical complexity and preservation methods.
- Despite difficulties, analyzing these specimens is crucial for understanding otological diseases.
Purpose of the Study:
- To develop and validate a method for extracting and analyzing mRNA from archival formalin-fixed, paraffin-embedded (FFPE) human inner ear tissues.
- To detect the expression of COCH and SLC26A5 mRNA in specific inner ear structures.
Main Methods:
- Human temporal bones were processed and embedded in paraffin.
- Laser microdissection was used to isolate specific tissues: spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis.
- RNA was extracted, heat-treated to remove modifications, and analyzed using semi-nested reverse transcription-polymerase chain reaction (RT-PCR).
Main Results:
- COCH mRNA was detected in the spiral ligament and stria vascularis.
- SLC26A5 mRNA was exclusively found in outer hair cells.
- COCH mRNA fragments up to 123 bp were successfully amplified from archival samples.
Conclusions:
- The study successfully detected COCH and SLC26A5 mRNA in distinct inner ear structures from archival human temporal bone.
- The developed method, combining laser microdissection and semi-nested RT-PCR, offers a promising approach for future RNA studies of human inner ear diseases.
