Itaq sybr green supermix pcr 1 master mix

MtDNA content was measured using quantitative real time polymer-ase chain reaction (qRT-PCR). RT-PCR reactions were performed via SYBR Green chemistry on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The primers were specific, respectively, for the rat mitochondrial D-loop region (numbering is according to GenBank™ accession number AY172581) and for the rat nuclear β-actin gene (numbering is according to GenBank™ accession number VO1217.1) and are listed in Table 1. The method has been validated by primer-limiting experiments and by evaluating the equal reaction efficiency of the two amplicons. Amplification specificity was controlled by melting curve analysis and gel electrophoresis. Each sample was analyzed in triplicate in 25 μl of final volume containing: iTaq SYBR Green Supermix PCR 1× Master Mix (Bio-Rad Laboratories Inc., Hercules, CA, USA), 0.2 μM forward and reverse primers, and DNA template (2.5 μl of diluted 1:10). After 10 min of denaturation at 95 °C, amplification proceeded for 40 cycles, each consisting of denaturation at 95 °C for 15 s, annealing, and extension at 60 °C for 1 min. The quantification of the relative mtDNA content in aged AL and CR rats, compared to young or middle-age rats, all normalized to β-actin, was performed according to the Pfaffl mathematical model [34 (link)].

The measurement of the relative content of mtDNA im- munoprecipitated by ΤFAΜ was carried out by qPCR. To analyze the D- loop, Ori-L, and ND1 mtDNA regions bound by TFAM, rt D-loop, rt Ori- L, and rt ND1 primer sets were used (Table 1). qPCR reactions were performed via SYBR Green chemistry on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and fluorescence spectra were monitored by the QuantStudio Real-Time PCR Software vl.3 (Applied Biosystems, Foster City, CA, USA). The reaction mixture (total volume 20 μL) consisted of iTaq SYBR Green Supermix PCR 1 × Master Mix (BioRad Laboratories Inc., Hercules, CA, USA), 0.2 μM forward and reverse primers, and 2.5 μL of the input or of the immunoprecipitated with anti-TFAM or without antibody DNA aliquots. After 10 min of denaturation at 95 ˚C, amplification proceeded for 40 cycles, each consisting of denaturation at 95 ˚C for 1 s, annealing and extension at 60 ˚C for 20 s (Fast block). The calculation of the relative content of TFAM-bound mtDNA was performed according to the formula 2^ΔCT× — 2^ΔCTb, where Δ^cT× is the difference between the CT values of the input and the immunoprecipitated sample and Δ^CTb is the CT difference between the CT values of the input and the no-antibody sample [21 (link)], respectively for each analyzed region.