Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue

Technical report: Laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR

“…To our knowledge, however, no studies exist which compare the integrity of DNA molecules isolated from human temporal bone tissues using 1% nitric acid, as is used in our laboratory, versus EDTA and our studies have not supported a concern. In this regard, we have recently demonstrated the presence of a native 10.4 kb mtDNA fragment using long range PCR and product sequencing in temporal bone tissue samples processed with formalin, ethanol, and nitric acid (Markaryan et al, 2008a). This finding confirms that the conditions used for processing the temporal bones in our laboratory preserve large fragments of mtDNA without base modification and suggests that the mtDNA present in these temporal bone specimens is a satisfactory source for the investigation of ear diseases.…”

“…A 417 bp mtDNA sequence of the D-loop was amplified from human blood by standard PCR using the following primers: forward -5 0 -CTA TCA CCC TAT TAA CCA CT (nt 11-30), reverse -GTT AAA AGT GCA TAC CGC CA (nt 427-408). A 316 bp sequence representing the CD was amplified from cochlear tissue as previously described (Markaryan et al, 2008a). Both fragments were cloned into the 3956-bp PCR4-TOPO vector (Invitrogen, Carlsbad, CA).…”

“…In addition, identification of mtDNA mutations was not attempted. Subsequently, Markaryan et al (2008a) have identified mtDNA deletions in cochlear elements isolated by LMD from archival human temporal bone tissues with presbycusis. This study utilized nested and long range PCR to identify three unique mtDNA deletions and the previously reported CD.…”

Gravity Receptor Aging in the CBA/CaJ Strain: A Comparison to Auditory Aging

“…ROMs are toxic molecules that can damage tissue and have been shown to damage mtDNA. The number of mtDNA mutations has been shown to be proportional to aging (Zhang et al 2002;Markaryan et al 2006) and as these mutations accumulate with advancing age, mitochondrial metabolic processes become progressively less efficient. Several studies have examined the role of mitochondria dysfunction in ARHL (e.g., Seidman 2000; Seidman et al 2000, 2002Fischel-Ghodsian et al 2004;Yamasoba et al 2007).…”

Antioxidants and Their Effect on Stress-Induced Pathology in the Inner Ear

“…Additional mtDNA defi cits have also been identifi ed which correlate with ARHL. Examples are the 5,142-bp and 5,354-bp deletions in mtDNA reported by Markaryan et al ( 2008 ). Finally, certain mtDNA haplogroups have been shown to be associated with ARHL (Manwaring et al 2007 ).…”