Ultrapure dnase rnase free h2o

The cfDNA concentrations were determined as described by Neuberger et al. [22 (link)]. Ahead of the measurements, the linearity, limit of quantification, and limit of detection of the assay were determined. The qPCR assay amplifies DNA in unpurified plasma, which is diluted 1:10 in UltraPure DNase/RNase-Free H2O (Invitrogen, Waltham, MA), targeting a 90 bp fragment of human long interspersing nuclear elements (LINEs) of the repetitive L1PA2 family (5′-TGCCGCAATAAACATACGTG-3′ and 5′-GACCCAGCCATCCCATTAC-3′). Briefly, each sample was measured as a technical triplicate with 5 µL final volume containing 0.66 µL of 1:10 diluted plasma, 0.33 µL primer mix (140 nm final concentration of each primer) and 4 µL of qPCR mix with 0.6 U Velocity Polymerase (Bioline, London, UK), 1.2 × Hifi Buffer (Bioline, London, UK), 0.1 × SYBR Green (Sigma, St. Louis, MO, USA), and 0.3 mM dNTPs (Bioline, London, UK). The qPCR reaction was carried out using a CFX384 Bio-Rad (Bio-Rad, Munich, Germany) cycler with a two-step protocol. The cycling conditions were: initial heat activation at 98 °C for 2 min followed by 33 cycles of 95 °C for 10 s and 64 °C for 10 s with a subsequent melting curve from 70 to 95 °C with 0.5 °C increments for 10 s. The operator measuring the samples was blinded and did not know the allocation of the samples into the control or intervention group.

Plasma samples were diluted 1:10 in UltraPure DNase/RNase-Free H₂O (Invitrogen, Waltham, MA). For the qPCR, 2 µl of the diluted plasma, were mixed with 12 µl master-mix, 1 µl primer mix, and measured in triplicates with a final volume of 5 µl. The primer sequences are listed in Supplementary Table S3. The final concentrations in the PCR reaction are 1.2 × HiFi buffer (BioCat, Heidelberg, Germany), 0.3 mM of each dNTP (Carl Roth, Karlsruhe, Germany), 0.15 × SYBR Green (Sigma-Aldrich, Taufkirchen, Germany), 0.04 IU Velocity Polymerase (BioCat, Heidelberg, Germany), and 140 nM of each primer. For amplification a Bio-Rad CFX384 system (Bio-Rad, Hercules, CA, USA), with the following protocol was used: 98 °C for 2 min followed by 35 cycles of 95 °C for 10 s (denaturation) and 64 °C for 10 s (annealing/extension) including the plate read. A melt curve from 70–95 °C with 0.5 °C increments for 10 s finished each run. If the triplicates of the samples showed a standard deviation (SD) of the quantification cycle (Cq) > 0.4, native plasma samples were re-diluted and reanalyzed.