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

X-ray tomography as a tool for detailed anatomical analysis

Wood identification, anatomical examination and retrieval of quantitative information arc important aspects of many research disciplines. Conventional light microscopy with a camera and (semi)automatic image analysis software is an often used methodology for these purposes. Morc advanced techniques such as fluorescence, scanning electron, transmission electron, confocal laser scanning and atomic force microscopy arc also part of the toolset answering to the need for detailed imaging. Fast, non-destructive visualization in three dimensions with high resolution combined with a broad field of view is sought-after, especially in combination with flexible software. A highly advanced supplement to the existing techniques, namely X-ray sub-micron tomography, meets these requirements. It enables the researcher to visualize the material with a voxel size approaching <1 mu m for small samples (<1 mm). Furthermore, with tailor-made processing software quantitative data about the wood in two and three dimensions can be obtained. Examples of visualization and analysis of four wood species arc given in this paper, focusing on the opportunities of tomography at micron and sub-micron resolution. X-ray computed tomography offers many possibilities for material research in general and wood science in specific, as a qualitative as well as a quantitative technique

Preventive action of organosilicon treatments against disfigurement of wood under laboratory and outdoor conditions

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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.JRC.I.3-Molecular Biology and Genomic

Verification Report on the extraction and analysis of GM pollen DNA in honey

Following the judgment of 06 September 2011 on GM honey by the European Court of Justice (legal case C-442-09), the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) established by Regulation (EC) No 1829/2003, performed an in-house study to test the extraction and PCR analysis of genomic DNA from genetically modified pollen in honey. The present report documents on an extraction method for isolation and analysis of pollen DNA present in honey, including the isolation and analysis of isolated genomic pollen DNA using real-time PCR on commercial honey samples and honey samples spiked with various levels of GM MON 810 pollen.JRC.I.3-Molecular Biology and Genomic

Fuzzy-logic based procedures for GMO Analysis

A monitoring system for GMOs (Genetically Modified Organisms) and GMO-derived products in the food and feed chains has been established in the European Union (EU) - . In previous letters to your journal - , challenges were highlighted to implement EU regulations for labeling of products containing GMOs. Test methods for monitoring GMOs are based mainly on the Polymerase Chain Reaction (PCR).JRC.I.3-Molecular Biology and Genomic

Fuzzy logic-based procedures for GMO analysis

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Technical Guidance Document from the European Union Reference Laboratory for Genetically Modified Food and Feed on the Implementation of Commission Regulation (EU) No 619/2011

The recently adopted Commission Regulation (EU) No 619/2011 lays down the methods for sampling and analysis for the official control of food as regards to presence of genetically modified material for which an authorisation procedure is pending or the authorisation of which is has expired (the so-called LLP, Low Level presence Regulation). The requirements of this Regulation are technically demanding because they set a non-compliance limit of 0.1% GMO (mass fraction). The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) plays a crucial role in the implementation of this regulation because only methods validated by the EU-RL that show a RSDr value of maximum 25% at the level of 0.1% related to mass fraction of GM material can be used on falling under that regulation. The aim of this document is to explain how this Regulation will affect the process of validation and how laboratories need to operate under that Regulation.JRC.I.3-Molecular Biology and Genomic

The role of vacuolar and secreted pathogenesis-related β (1-3)-glucanases and chitinases in the defence response of plants

Upon infection of a plant by a pathogen, a series of drastic metabolic changes occur within the plant. One characteristic feature of this defence response is the synthesis of the so-called pathogenesis-related (PR) proteins. We have studied the nature, structure, and subcellular localization of the different PR proteins upon salicylic acid treatment and Pseudomonas syringae infection of Nicotiana tabacum plants. In both test systems, we could demonstrate that the PR protein fraction of tobacco consists of at least 20 to 25 different proteins, including beta (1-3)-glucanases, chitinases, peroxidases, thaumatin-like proteins, the PR1 class proteins and a proteinase inhibitor-like protein. Moreover, several classes of these PR proteins segregate into specific vacuolar and secreted isoforms. Here, we present a model which could explain the role of the compartimentalized PR beta (1-3)-glucanases and chitinases within the regulation of the defence response