Accumulation of mtDNA common deletion in the dermis of naturally aged and photoaged human skin in vivo.
a Schematic representation of the dissection of human skin dermis and detection of mtDNA common deletion in the dermis. a) Haematoxylin & Eosin staining of full-thickness human skin; b) Skin dermis was prepared by cutting off epidermis at a depth of 1 mm by cryostat; c) Purified total DNA from the dermis; d) Detection of mtDNA common deletion in the dermis. Real-time QPCR primers A, B, and C (indicated by arrows) were used to simultaneously PCR DNA fragments from both wild-type mtDNA (primers A/B) and deleted mtDNA (primers A/C). The PCR fragment from primers A/C was too large for wildtype mtDNA (>5 kb) to be amplified with the PCR conditions used for detection (see Methods for details). b Minimal contamination of keratinocyte in the dermis. Total RNA was prepared from epidermal and dermal sections, and keratin 14 (marker of epidermis) and type I procollagen (marker of dermal fibroblasts) mRNA levels were quantified by real-time RT-PCR. Keratin 14 and type I procollagen mRNA levels were normalized by the housekeeping gene (36B4, internal control). Mean ± SEM. N = 6, *p < 0.05. c mtDNA common deletion is increased in the dermis of naturally aged human skin in vivo. mtDNA common deletion was determined by real-time QPCR (see details for Methods). Young group (N = 8, mean age 24 ± 3 years) and aged group (N = 8, mean age 82 ± 4 years). Mean ± SEM. *p < 0.05. d mtDNA common deletion is increased in the dermis of photoaged human skin in vivo. mtDNA common deletion was determined by real-time QPCR (see Methods for details). Photoaged skin and subject matched sun-protected skin were obtained from the extensor forearm and sun-protected buttocks, respectively. Mean ± SEM. N = 10. *p < 0.05