Muscle samples were incubated overnight in 360 μL of buffer proprietary
ATL lysis buffer and 40 μL proteinase (Qiagen) at 55°C in a thermomixer set at 300 r.p.m. Cell debris was removed by centrifugation and 200 μL of clear lysates was placed in the
QIAsymphony SP workstation (Qiagen). DNA was extracted with the
QIAsymphony DNA Mini kit (Qiagen, catalogue no. 937236) following the manufacturer’s procedures. Quantitative PCR was performed on the Fluidigm Biomark system following the
Fluidigm Specific Target Amplification Quick Reference (Fluidigm). Samples were loaded as technical triplicates. The real-time PCR data were analysed using the Linear Derivative baseline correction and User (detector) Ct threshold method on the latest version of the Fluidigm Biomark software (v.4.1.3). Quantification of mtDNA was performed using two customized Taqman assays targeted against a nuDNA sequence (18S) and a conserved region of mtDNA (MTND1). Relative mtDNA copy number was determined comparing MTND1 to 18S signal. qPCR data quality control was performed and samples with low DNA sample concentration or low sample purity were discarded. These criteria were defined before starting the analysis of the raw data. Sample belonging to 4 subjects (2 placebo and 2 UA 1000 mg) did not meet the QC requirements and were excluded from the analysis
Singh A., D’Amico D., Andreux P.A., Fouassier A.M., Blanco-Bose W., Evans M., Aebischer P., Auwerx J, & Rinsch C. (2022). Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults. Cell Reports Medicine, 3(5), 100633.
