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Duplex Real-Time PCR Assay for Quantifying Mitochondrial DNA Deletions in Laser Microdissected Single Spiral Ganglion Cells

  • Adam MarkaryanEmail author
  • Erik G. Nelson
  • Raul Hinojosa
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 755)

Abstract

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this chapter, we describe a technique for isolating individual spiral ganglion cells from archival formalin-fixed, celloidin-embedded (FFCE) human temporal bone sections. The DNA isolated from these single cells is suitable for analysis with a duplex real-time polymerase chain reaction (PCR) methodology to quantify the mitochondrial DNA (mtDNA) deletion level present.

Key words

Presbycusis Human temporal bones Mitochondrial DNA deletions Duplex real-time PCR assay Laser microdissection 

Notes

Acknowledgments

This work was supported by the Bloom Temporal Bone Laboratory Endowment and a 2009 Deafness Research Foundation Grant provided by the Burch-Safford Foundation to A.M.

References

  1. 1.
    Reeve AK, Krishnan KJ, Turnbull D (2008) Mitochondrial DNA mutations in disease, aging and neurodegeneration. Ann NY Acad Sci 1147:21–29.CrossRefGoogle Scholar
  2. 2.
    Markaryan A, Nelson EG, Hinojosa R (2008) Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue. Mutation Research 640:38–45.CrossRefGoogle Scholar
  3. 3.
    Markaryan A, Nelson EG, Hinojosa R (2009) Quantification of the mitochondrial DNA common deletion in presbycusis. Laryngoscope 119:1184–9.CrossRefGoogle Scholar
  4. 4.
    Markaryan A, Nelson EG, Hinojosa R (2010) Major arc mitochondrial DNA deletions in cytochrome c oxidase-deficient human cochlear spiral ganglion cells. Acta Oto-Laryngologica 130:780–787.Google Scholar
  5. 5.
    Seidman MD, Bai U, Khan MJ, Murphy MP, Quirk WS, Castora FJ, Hinojosa R (1996) Association of Mitochondrial DNA Deletions and Cochlear Pathology: A Molecular Biology Tool. Laryngoscope 106:777–783.CrossRefGoogle Scholar
  6. 6.
    Markaryan A, Nelson EG, Tretiakova M, Hinojosa R (2008) Laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR. Hear Res 244:1–6.CrossRefGoogle Scholar
  7. 7.
    Nelson EG, Hinojosa R (2003) Presbycusis: A human temporal bone study of individuals with flat audiometric patterns of hearing loss using a new method to quantify stria vascularis volume. Laryngoscope 113:1672–86.CrossRefGoogle Scholar
  8. 8.
    Nelson EG, Hinojosa R (2006) Presbycusis: A human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature. Laryngoscope 116 (Suppl 112):1–12.CrossRefGoogle Scholar
  9. 9.
    Sabunciyan S, Kirches E, Krause G, Bogerts B, Mawrin C, Llenos IC, Weis S (2007) Quantification of total mitochondrial DNA and mitochondrial common deletion in the frontal cortex of patients with schizophrenia and bipolar disorder. J Neural Transm 114:665–674.CrossRefGoogle Scholar
  10. 10.
    He L, Chinnery PF, Durham SE, Blakely EL, Wardell TM, Borthwick GM, Taylor RW, Turnbull DM (2002) Detection and quantification of mitochondrial DNA deletions in individual cells by real- time PCR. Nucleic Acid Research 30(e68):1–6.Google Scholar
  11. 11.
    Yin S, Yu Z, Sockalingam R, Bance M, Sun G, Wang J (2007) The role of mitochondrial DNA large deletion for the development of presbycusis in Fischer 344 rats. Neurobiol Dis 27:370–377.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Adam Markaryan
    • 1
    Email author
  • Erik G. Nelson
    • 1
  • Raul Hinojosa
    • 1
  1. 1.Section of Otolaryngology – Head and Neck Surgery, Department of SurgeryUniversity of ChicagoChicagoUSA

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