LORD-Q assay

Measurement of mitochondrial and nuclear DNA damage was performed by the LORD-Q method as originally described [12]. For each analyzed genomic or mitochondrial gene locus a long DNA fragment (~3000 – 4000 bp) was used as sensor for DNA lesions and an internally nested short fragment (~50 – 70 bp) that was considered as undamaged served as reference. The nested intact reference was efficiently PCR-amplified, whereas amplification of the large sensor is inhibited by DNA lesions including abasic sites, thymine dimers, strand breaks and oxidative lesions [12]. Upon induction of DNA damage, the exponential amplification phase for the damage-sensitive large fragment is reached later as compared to the nested intact reference. The difference in crossing point values (C_p) therefore allows calculation of the average incidence of lesions per bp.

Initially, the replication efficiency of each primer pair was determined using a standard dilution of whole-cell DNA as described [12]. Then, real-time PCR was carried out in 96-well or 384-well plates with each well containing a reaction volume of 20 μL and 10 μL, respectively. The reaction mixture contained 2.5 ng/μL isolated sample DNA, 1 x KAPA2G HS Polymerase ReadyMix (Peqlab), 0.0016 x ResoLight dye (Roche, Basel, Switzerland) and 500 nM of HPLC-purified forward and reverse primers (Sigma-Aldrich), respectively.

Cycling conditions were as follows: a pre-incubation phase of 5 min at 95°C was followed by up to 60 cycles of 10 s at 95°C, 10 s at 60°C and 1 s at 72°C for small amplicons or 135 s for large amplicons, respectively. The reactions were carried out in a LightCycler 480 II system (Roche). Samples were measured in triplicates (96-well plates) or quadruplicates (384-well plates). C_p values were calculated using the LightCycler 480 software and replicates were averaged. All primers were designed to match the requirements of LORD-Q and are listed in Supplementary Table 1. For detection of nuclear DNA damage promoter sequences of the human and murine COL1A1 locus were analyzed. Data is depicted as mean ± standard deviation from at least three independent experiments.