Easy dna isolation kit

Total DNA was isolated from DRG neurons (3×10⁴) using easy DNA isolation kit (Life Technologies) and RNA was digested with RNase A (40 μg/ml/30 min/37°C). Real-time qPCR reactions were assembled in duplicates with SSO FAST Evagreen supermix (Biorad, Hercules, CA). The single-copy nuclear gene beta-2-microglobulin (B2M) was used as reference for determination of mtDNA copy number. A 180-nucleotide fragment of mitochondrial gene encoding cytochrome oxidase subunit III (Cox III) served amplification target after validation of qPCR data with two additional mitochondria encoded genes, Cox1 and ND1 that yielded similar results. mtDNA copy number was calculated using the formula: mtDNA copy number =2×2^{(CTCoxIII − CTB2M)} [51 (link),52 (link)]. Primers for CoxIII (MGI:102502) were F-caattacatgagctcatcatagc and R-ccatggaatccagtagcca and for B2M (NM_009735) F-atccaaatgctgaagaacgg and R-atcagtctcagtgggggtga. Three sets of independent experiments were carried out and mean±SEM values for mtDNA copy number were calculated.

Total DNA was isolated from ~3×10⁴ DRG neurons using easy DNA isolation kit (Life Technologies) and RNA was digested with RNase A (40 µg/ml/30 min/37°C). Real-time qPCR reactions were assembled in duplicates with 10 ng total DNA and SSO FAST Evagreen supermix (Biorad, Hercules, CA). The single-copy nuclear gene beta-2-microglobulin (B2M) was used as reference for determination of mtDNA copy number. A 180-nucleotide fragment of mitochondrial gene encoding cytochrome oxidase subunit 3 (COX3) was amplified after validation of qPCR data by targeting two additional mitochondria encoded genes, COX1 and ND1 that yielded similar results. mtDNA copy number was calculated using the formula: mtDNA copy number =2×2^{(CTCoxIII − CTB2M)} [28 (link), 35 (link), 36 ]. Primers for COX3 (ID 17710) were F-caattacatgagctcatcatagc and R-ccatggaatccagtagcca and for B2M (NM_009735) F-atccaaatgctgaagaacgg and R-atcagtctcagtgggggtga. Three sets of independent experiments were carried out and mean±SEM values for mtDNA copy number were calculated.