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.

Development and application of a real-time TaqMan((R)) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan((R)) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.

Selecting control genes for RT-QPCR using public microarray data

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. RESULTS: We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at http://www.isrec.isb-sib.ch/~vpopovic/research/ CONCLUSION: We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.

Aktiensplits vor der Tür

Eine typische Schweizer Aktie kostete Anfang März 2001 über CHF 1000. Das ist ein Weltrekord. In keinem Land liegen die Aktienkurse auch nur annähernd so hoch. Mit einer Gesetzesänderung wird der Mindestnennwert per 1. Mai 2001 von CHF 10 auf 1 Rp. herabgesetzt. Dies ermöglicht Schweizer Gesellschaften, die Kurse ihrer Aktien durch Splits auf international übliche Werte zu vermindern. Wie verbreitet ist dieses Bedürfnis? Welche Auswirkungen haben Aktiensplits auf die Börsenbewertung? Welche Vorteile sind damit verbunden? Was ist der «optimale» Börsenkurs? Welche Gesellschaften sind primäre Splitkandidaten? Antworten auf diese Fragen finden sich im vorliegenden Aufsatz.

Prostitúciós terek performativitása - szexmunkások és a nyilvános tér Magyarországon

Az állami intézmények tereket hoznak létre azáltal, hogy meghatározott területekre vonatkozóan szabályokat vagy normákat írnak elő. Az ott élő, tevékenykedő emberek használják e tereket, stratégiákat alkotnak, alkalmazkodnak a törvényekhez és piaci viszonyokhoz, kiskapukat keresnek, hogy megélhetésüket, túlélésüket biztosítsák. Eközben ők maguk is olyan tereket hoznak létre, amelyeket a társadalom szimbolikusan értékel vagy stigmatizál. De a terek maguk is alakítják az embereket, lehetőséget biztosítanak számukra, hogy alkalmazkodjanak a feltételekhez, amivel ugyanakkor megteremtik a társadalmi változások alapjait – akárhogyan is alakuljanak e változások. A médiában, a prostitúcióról szóló hírekben a főszereplők jellemzően háttérben maradnak, e tanulmány nekik szentel figyelmet. Ezzel egyidejűleg a szerző megkíséreli a magyarországi nyilvános tereken zajló prostitúciót Budapest, Pécs és Nyíregyháza legkülönfélébb terein megvizsgálni.

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.