- 作者：Yurika Kimura ; Sachiho Kubo ; Hiroko Koda ; Kazuhiro Shigemoto ; Motoji Sawabe ; Ken Kitamura
- 关键词：FFPE ; formalin fixed paraffin embedded ; EDTA ; ethylenediaminetetraacetic acid ; mtDNA ; mitochondrial DNA ; LMD ; laser microdissection ; SV ; stria vascularis ; SGC ; spiral ganglion cells ; OHC ; outer hair cells ; SL ; spiral ligament
- 刊名：Hearing Research
- 出版年：2013
- 出版时间：August, 2013
- 年：2013
- 卷：302
- 期：Complete
- 页码：26-31
- 全文大小：1250 K
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.