Scai digestion

In order to verify whether the two fluorescent dyes present different intensities of fluorescence emission and to normalize for differences between runs (master mix performance, instrument calibration, environmental variability, etc.) a plasmid construct was designed. A portion of mt-tRNA^Leu and B2M genes were amplified using the previously reported primers. Two sequential TA cloning reactions were performed to insert the two targets into the pGEM-T vector (Promega Co., Madison, WI, USA). The successful cloning of the two target sequences in a 1:1 ratio was verified by sequencing and PCR. The distance between both inserts was chosen to be greater than 500 bp to avoid the amplification of both targets as one amplicon in the qPCR reaction (Fig. 1).Figure 1

Scheme of the linearized dual insert plasmid (pGEM-T vector, 3194 bp) showing the inserts of human mtDNA and nuclear DNA.

Plasmids were extracted from NEB Turbo Competent E. coli (New England Biolabs, Ipswich, MA, USA) with PureYield Plasmid Midiprep kit (Promega Co., Madison, WI, USA) and the concentration was measured using Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The plasmid was linearized by ScaI digestion (New England Biolabs, Ipswich, MA, USA) and the linearized plasmid was used as calibrator in all the qPCR experiments.