qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Fragmentation with a Cut on Thrust: Predictions for B-factories

When high-energy single-hadron production takes place inside an identified jet, there are important correlations between the fragmentation and phase-space cuts. For example, when one-hadron yields are measured in on-resonance B-factory data, a cut on the thrust event shape T is required to remove the large b-quark contribution. This leads to a dijet final-state restriction for the light-quark fragmentation process. Here, we complete our analysis of unpolarized fragmentation of (light) quarks and gluons to a light hadron h with energy fraction z in e+e−→dijet+h at the center-of-mass energy Q=10.58  GeV. In addition to the next-to-next-to-leading order resummation of the logarithms of 1−T, we include the next-to-leading order nonsingular