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Quantitative analysis of mRNA in human temporal bones
Yurika Kimura1, Sachiho Kubo, Hiroko Koda
1Department of Otolaryngology, Tokyo Metropolitan Geriatric Hospital, Japan.
Conclusion:
Well-preserved mRNA could be extracted from frozen human inner ears. Therefore, this study demonstrates that analysis of mRNA could be performed to study the molecular mechanisms of inner ear disorders using human specimens.
Objectives:
Analysis of RNA as well DNA is requisite to study the molecular mechanisms of inner ear disorders. Methods of isolating RNA from experimental animals have been established, while isolation of RNA from human inner ears is much more challenging. In the present study, we demonstrate a method by which messenger RNA (mRNA) was extracted from human inner ears and quantitatively analyzed.
Materials And Methods:
COCH mRNA as well as GAPDH mRNA was extracted from membranous labyrinths dissected from three formalin-fixed and three frozen human temporal bones, removed at autopsy. The length of COCH mRNA and quantity of GAPDH mRNA was compared between the two groups by quantitative RT-PCR.
Results:
COCH mRNA could be amplified as much as 976 bp in all three frozen specimens. By contrast, it was amplified to 249 bp in two of the three formalin-fixed specimens, with no amplification observed in the remaining. The quantity of amplifiable GAPDH mRNA in the formalin specimens was only 1% of that of the frozen specimens.
Insights
Frozen human inner ear tissue yields well-preserved messenger RNA (mRNA), enabling molecular studies of inner ear disorders. This method is crucial for understanding disease mechanisms in human specimens.
Area of Science:
- Otolaryngology
- Molecular Biology
- Human Genetics
Background:
- Studying the molecular mechanisms of inner ear disorders requires RNA and DNA analysis.
- Established methods for RNA isolation exist for animal models, but human inner ear RNA isolation is challenging.
Purpose of the Study:
- To demonstrate a method for extracting and quantitatively analyzing messenger RNA (mRNA) from human inner ears.
- To establish a protocol for molecular analysis of human inner ear tissues.
Main Methods:
- Messenger RNA (mRNA) was extracted from human inner ear tissues (membranous labyrinths) obtained from both frozen and formalin-fixed temporal bones.
- Quantitative RT-PCR was used to compare the length and quantity of COCH mRNA and GAPDH mRNA between frozen and formalin-fixed specimens.
Main Results:
- Well-preserved COCH mRNA (up to 976 bp) was successfully amplified from all three frozen specimens.
- Formalin-fixed specimens yielded significantly less amplifiable COCH mRNA (249 bp) and only 1% of the GAPDH mRNA quantity compared to frozen specimens.
Conclusions:
- Frozen human inner ear tissue is suitable for extracting well-preserved mRNA.
- This method allows for the molecular study of inner ear disorders using human specimens.
