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ana23955-sup-0001-suppfig1.eps439KSupplementary Fig 1. Ultrafiltration removes PCR inhibitory factors from CSF. Real time qPCR amplification curves of mtDNA-85 (A), mtDNA-153 (B) and mtDNA-515 (C) performed either in a calibration standard of purified human mtDNA (100 copies) from HEK293T cells (STANDARD), directly in CSF (CSF) or in ultrafiltrated CSF (UF CSF). Direct mtDNA amplification in CSF samples results in an amplification curve with a significantly smaller slope than the obtained in the human mtDNA calibration standard, indicating the presence of PCR inhibitors that reduce PCR reaction efficiency and compromise accurate quantification. Ultrafiltration of the CSF samples with 10,000 MWCO Amicon Ultra 0,5ml filters completely removed PCR inhibitors yielding amplification curve slopes and PCR reaction efficiencies equivalent to the observed in the mtDNA standard for all the mtDNA amplicons. Quantitative analyses of the slopes of PCR amplification curves, expressed as number of cycles between 20 and 80 relative fluorescence unit thresholds (Cq80-Cq20), are represented in the graphs on the right for each of the mtDNA amplicons. Values are mean ±SEM of relative fluorescence units (left panel) or PCR cycles between threshold 20 and 80 (right panel) of n=3 independent real time PCR runs. **, p<0.01, significantly different from the corresponding HEK293T standard (Oneway ANOVA with Bonferroni post-hoc comparisons).
ana23955-sup-0002-suppfig2.eps350KSupplementary Fig 2: Gender analysis of mtDNA content in CSF. Global (A) and within groups (B) gender analysis of the mtDNA content in CSF measured with three different primer combinations in samples from the study cohort shown in Fig 1C. Values are expressed in mean±SEM of fg/ml. (C) Global gender analysis of the mtDNA content in CSF measured with the mtDNA-73 primer combination in samples from the validation cohort. The number above bars represents the number of subjects per group.
ana23955-sup-0003-supptable1.doc107KSupplementary Table 1 Clinical and biomarker individual values of study subjects analyzed by Real Time qPCR
ana23955-sup-0004-supptable2.doc72KSupplementary Table 2 Clinical and biomarker individual values of study subjects analyzed by digital droplet PCR
ana23955-sup-0005-supptable3.doc92KSupplementary Table 3 Clinical and biomarker individual values of study subjects from the confirmatory cohort analyzed by Real Time qPCR

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