title: RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Department of Otolaryngology, Tokyo Metropolitan Geriatric Medical Hospital, 35-2, Itabashi, Tokyo 173 0015, Japan. kimura@tmghig.jp
authors: Yurika
authors: Y
authors: Kimura
authors: Sachiho
authors: S
authors: Kubo
authors: Hiroko
authors: H
authors: Koda
authors: Kazuhiro
authors: K
authors: Shigemoto
authors: Motoji
authors: M
authors: Sawabe
authors: Ken
authors: K
authors: Kitamura
mesh_terms: D000368:Aged; D027321:Anion Transport Proteins / Q000378:metabolism; D002102:Cadaver; D018076:DNA, Complementary / Q000032:analysis; D007758:Ear, Inner / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D005557:Formaldehyde; D005786:Gene Expression Regulation; D006198:Hair Cells, Auditory / Q000378:metabolism*; D006801:Humans; D061346:Laser Capture Microdissection; D008297:Male; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D000076988:Sulfate Transporters; D013701:Temporal Bone / Q000378:metabolism*; D014021:Tissue Preservation
chemical_list: D027321:Anion Transport Proteins; C110668:COCH protein, human; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; D005557:Formaldehyde