Figure 6. Interrogation and characterization of exosome fraction DNA using mtDNA and NUMT-directed primers.

(A) DNA isolated from NT2 cells, NT2 ρ0 cells, NT2 cell-derived exosomes, and NT2 ρ0 cell-derived exosomes, with and without DNase I-treatment, was amplified with primers to the mtDNA ND2 gene. The products of this PCR reaction were then digested with Hph I. Restriction of the ND2 band indicates 5460A, which is found in authentic mtDNA. Absence of ND2 band restriction indicates 5460G, which is not present in NT2 mtDNA and is found in at least one ND2 NUMT. (B) DNA isolated from NT2 cells, NT2 ρ0 cells, and NT2 cell-derived exosomes treated or not treated with DNase I was first amplified with primers targeted to just beyond the borders of the Herrnstadt et al. NUMT (Numt_exclusive primers), or just within the boundaries of the NUMT (Numt_inclusive primers). Following these PCR amplifications aliquots were taken and subjected to PCR amplification with primers to ND2. The ND2 amplicons were then digested with Hph I. Data shown are representative of three experiments.