Workshop report on the extraction of foetal DNA from maternal plasma

Objective Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed. Methods Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration. Results The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with ≥ 500 µL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3. Conclusions This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies. Copyright © 2007 John Wiley & Sons, Ltd.     

Quantitative PCR to estimate copepod feeding

Copepods play a central role in marine food webs as grazers of plankton and as key prey for many predators. Therefore, quantifying their specific trophic interactions is critical for understanding the role of copepods in ocean processes. However, because of methodological constraints, it remains difficult to investigate in situ copepod feeding without reliance on laborious intrusive and potentially biased incubation approaches. Recent advances in PCR-based methodologies have demonstrated the feasibility of directly identifying copepod diets based on prey DNA sequences. Yet, obtaining quantitative information from these approaches remains challenging. This study presents results of systematic efforts to develop a quantitative PCR (qPCR) assay targeted to 18S rRNA gene fragments to estimate copepod gut content of specific species of prey algae. These results were first compared to gut content estimates based on fluorescence in the copepod Calanus finmarchicus fed monocultures of two different microalgae species in controlled laboratory studies. In subsequent field studies, we compared feeding rates obtained by microscopy and qPCR for Temora longicornis and Acartia clausi feeding on the haptophyte Phaeocystis globosa in natural blooms. These investigations demonstrate a semi-quantitative relationship between gut content estimates derived from qPCR, gut pigment, and direct microscopy. However, absolute estimates of gut content based on qPCR methodology were consistently lower than expected. This did not appear to be explained by the extraction methods used, or interference by non-target (predator) DNA in the PCR reactions, instead suggesting digestion of prey-specific nucleic acids. Furthermore, the 18S rDNA target gene copy number of the phytoplankton varied with growth phase. Nonetheless, when prey target gene copy number in the ambient water is quantified, the qPCR-approach can be compared to other methods, and then used to semi-quantitatively estimate relative copepod grazing on specific prey in situ without involving further incubations. A distinct advantage of a DNA-based molecular approach compared to gut fluorescence and direct microscopic observation, is the ability to detect non-pigmented and macerated prey. Future studies should aim to correct for breakdown in prey DNA and perform extensive calibrations to other methods in order to achieve a quantitative measure of feeding rates in situ.     

Mutual tolerance or reproductive competition? Patterns of reproductive skew among male redfronted lemurs (<Emphasis Type="Italic">Eulemur fulvus rufus</Emphasis>)

The social organization of gregarious lemurs significantly deviates from predictions of the socioecological model, as they form small groups in which the number of males approximately equals the number of females. This study uses models of reproductive skew theory as a new approach to explain this unusual group composition, in particular the high number of males, in a representative of these lemurs, the redfronted lemur (Eulemur fulvus rufus). We tested two central predictions of “concession” models of reproductive skew theory, which assume that subordinates may be allowed limited reproduction by dominant group members as an incentive to remain in the group, thereby increasing the group’s overall productivity. Accordingly, relatives are predicted to receive less reproduction than non-relatives, and the overall amount of reproductive concessions given to subordinates is predicted to increase as the number of subordinates increases. In addition, we tested whether the number of females in a group, a variable not previously incorporated in reproductive skew theory, affected reproductive skew among males. Using microsatellite analyses of tissue DNA, we determined paternities of 49 offspring born into our study population in Kirindy forest (western Madagascar) since 1996 to determine patterns of male reproductive skew to test these predictions. Our analyses revealed remarkable reproductive skew, with 71% of all infants being sired by dominant males, but both predictions of reproductive skew models could not be supported. Instead, the number of females best predicted the apportionment of reproduction among the males in this species, suggesting that current reproductive skew models need to incorporate this factor to predict reproductive partitioning among male primates and perhaps other group-living mammals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Both Peter M. Kappeler and Markus Port contributed equally to this paper.     

The influence of south Foehn on the ozone distribution in the Alpine Rhine valley—results from the MAP field phase

During the Mesoscale Alpine Programme (MAP) special observation period (SOP) between 7 September and 15 November 1999, ground-based and airborne measurements have been conducted in the Rhine valley south of the Lake of Constance to investigate the unstationary aspects of Foehn and related phenomena, like the impact of Foehn on the ozone concentrations in the valley. Foehn events occurred with above-average frequency and high diversity. Foehn induced ozone peaks in October and November are found to be much lower than the September Foehn case of the period. An inversion layer in the lake area with ozone concentrations below 10 ppb often shields the monitoring stations from the Foehn air aloft. Trajectory calculations for the Foehn period between 19 and 24 October 1999 reveal that the Foehn air originated from below 1 to 1.5 km above the Po Basin and the Mediterranean Sea. Tethered balloon soundings in the source area south of the Alps, ozone measurements at the mountain station Jungfraujoch (3580 m a.s.l.) and airborne measurements across the Alpine crests reveal that the ozone levels found in the Foehn air correspond to the concentrations just above the mixing height in the Po Basin and are transported across the Alpine crest within the lowest flow layer.