Enhanced analysis of real-time PCR data by using a variable efficiency model : FPK-PCR

Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a 'window of application', one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided

Simulation of between repeat variability in real time PCR reactions

While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in C-q values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats' plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond

3D Experimental investigation of the hygro-mechanical behaviour of wood at cellular and sub-cellular scale: detection of local deformations

The swelling/shrinkage of spruce wood samples (Picea Abies) is documented with high resolution XRay Tomography and advanced image analysis tools. We report the reversible moisture-induced global and local deformations at the cellular and sub-cellular scales. In particular, we present sophisticated methods for detecting local deformations in the cell wall. Insight is given on the hygromechanical behaviour of wood cell material and on the role of ultra-cellular components in wood, such as bordered pits and rays

In situ, in vitro and in silico analysis of coating performance

Service life prediction (SLP) is a complex yet essential method for analysing coating performance. Accurate and precise assessment is crucial for a strong position on the market. Correct knowledge of the critical failure points, maintenance/replacement frequency and related costs are part of this approach. This paper describes the scale-dependent tools within a bio-engineering framework that are applied for coating research, ranging from modelling, X-ray tomography, lab testing, controlled fungal infestation up to continuous moisture set-ups and large-scale window frame weathering