The m.7486G>A mt-tRNA mutation load was assessed by pyrosequencing technology, in all available tissues and in individual COX-positive and COX-deficient muscle fibres. The PyroMark Assay Design Software v.2.0. (Qiagen) was used to design locus-specific PCR and sequencing primers for the m.7486G>A variant (biotinylated forward primer: m.7466–7485; reverse primer: m.7583–7600; sequence primer: m.7488–7502) and pyrosequencing was performed on the Pyromark Q24 platform, according to the manufacturer's protocol. Pyromark Q24 software was used to quantify the m.7486G>A heteroplasmy levels by directly comparing the peak heights of both wild-type and mutant nucleotides at this position [42] (link).
The multiplex MTND1/MTND4 real-time PCR assay was performed using DNA from individual COX-deficient and COX-positive isolated muscle fibres and muscle homogenate and the mtDNA deletion level was calculated from the proportion of wild-type (MTND4) to total (MTND1) copy number by the established ΔΔCt method [43] (link). PCR amplification was completed in a 25 µl reaction in triplicate for each sample, with each plate containing a serial dilution of p7D1 plasmid for standard curve generation, as reported previously [44] (link).
The multiplex MTND1/MTND4 real-time PCR assay was performed using DNA from individual COX-deficient and COX-positive isolated muscle fibres and muscle homogenate and the mtDNA deletion level was calculated from the proportion of wild-type (MTND4) to total (MTND1) copy number by the established ΔΔCt method [43] (link). PCR amplification was completed in a 25 µl reaction in triplicate for each sample, with each plate containing a serial dilution of p7D1 plasmid for standard curve generation, as reported previously [44] (link).
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