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Biomolecular Detection and Quantification

Somanath Bhat, Kerry R Emslie
Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard...
December 2016: Biomolecular Detection and Quantification
J Madic, A Zocevic, V Senlis, E Fradet, B Andre, S Muller, R Dangla, M E Droniou
Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis...
December 2016: Biomolecular Detection and Quantification
Gerwyn M Jones, Eloise Busby, Jeremy A Garson, Paul R Grant, Eleni Nastouli, Alison S Devonshire, Alexandra S Whale
Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR...
December 2016: Biomolecular Detection and Quantification
Pawel R Debski, Piotr Garstecki
We explain how to design classic digital assays, comprising identical partitions, in order to obtain the required precision of the estimate within a defined range of concentrations. The design, including the number and volume of partitions, depends significantly on whether the assay is to assess the concentration of the target analyte in the sample or in the source of the sample (e.g. a patient body) with a given precision. We also show how to translate the result referring to the concentration in the sample into the concentration in the source of the sample, including the significant change in the breath of the confidence intervals...
December 2016: Biomolecular Detection and Quantification
Alexandra S Whale, Jim F Huggett, Svilen Tzonev
Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction...
December 2016: Biomolecular Detection and Quantification
Matthew E R Butchbach
Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes...
December 2016: Biomolecular Detection and Quantification
Harvinder S Dhillon, Gemma Johnson, Mark Shannon, Christina Greenwood, Doug Roberts, Stephen Bustin
BACKGROUND: The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest...
December 2016: Biomolecular Detection and Quantification
Valerie Taly, Jim Huggett
No abstract text is available yet for this article.
December 2016: Biomolecular Detection and Quantification
Stephen Bustin
No abstract text is available yet for this article.
September 2016: Biomolecular Detection and Quantification
Liesbet Deprez, Philippe Corbisier, Anne-Marie Kortekaas, Stéphane Mazoua, Roxana Beaz Hidalgo, Stefanie Trapmann, Hendrik Emons
Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented...
September 2016: Biomolecular Detection and Quantification
Fran Van Heuverswyn, Maria Karczmarczyk, Heinz Schimmel, Stefanie Trapmann, Hendrik Emons
Compared to other PCR technologies, digital PCR is a potentially highly accurate approach for the quantification of nucleic acid fragments. This study describes the impact of four experimental factors, namely primer and probe chemistry, PCR amplification target, duplexing, and template type, on the measurement results obtained by reverse transcription digital PCR (RT-dPCR) of viral RNA using influenza A virus as a model. Along conventional dual labelled probes (DLP), alternative primer and probe chemistries, including Zip Nucleic Acids (ZNAs), Locked Nucleic Acids (LNAs), and Scorpions(®), were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (In vitro transcribed RNA)...
September 2016: Biomolecular Detection and Quantification
Michał Burdukiewicz, Stefan Rödiger, Piotr Sobczyk, Mario Menschikowski, Peter Schierack, Paweł Mackiewicz
The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments...
September 2016: Biomolecular Detection and Quantification
Matthijs Vynck, Jo Vandesompele, Nele Nijs, Björn Menten, Ariane De Ganck, Olivier Thas
The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability. We propose the generalized linear mixed models framework as a flexible tool for analyzing a wide range of experiments. We also introduce a method for estimating reference gene stability to improve accuracy and precision of copy number and relative expression estimates...
September 2016: Biomolecular Detection and Quantification
Ian A Cree
RAS mutation analysis is an important companion diagnostic test. Treatment of colorectal cancer with anti-Epidermal Growth Factor Receptor (EGFR) therapy requires demonstration of RAS mutation status (both KRAS and NRAS), and it is good practice to include BRAF. In Non-Small Cell Lung Cancer (NSCLC) and melanoma, assessment of RAS mutation status can be helpful in triaging patient samples for more extensive testing. This mini-review will discuss the role of PCR methods in providing rapid diagnostic information for cancer patients...
June 2016: Biomolecular Detection and Quantification
Alison S Devonshire, Rebecca Sanders, Alexandra S Whale, Gavin J Nixon, Simon Cowen, Stephen L R Ellison, Helen Parkes, P Scott Pine, Marc Salit, Jennifer McDaniel, Sarah Munro, Steve Lund, Satoko Matsukura, Yuji Sekiguchi, Mamoru Kawaharasaki, José Mauro Granjeiro, Priscila Falagan-Lotsch, Antonio Marcos Saraiva, Paulo Couto, Inchul Yang, Hyerim Kwon, Sang-Ryoul Park, Tina Demšar, Jana Žel, Andrej Blejec, Mojca Milavec, Lianhua Dong, Ling Zhang, Zhiwei Sui, Jing Wang, Duangkamol Viroonudomphol, Chaiwat Prawettongsopon, Lina Partis, Anna Baoutina, Kerry Emslie, Akiko Takatsu, Sema Akyurek, Muslum Akgoz, Maxim Vonsky, L A Konopelko, Edna Matus Cundapi, Melina Pérez Urquiza, Jim F Huggett, Carole A Foy
Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA...
June 2016: Biomolecular Detection and Quantification
Kirill Kondratov, Dmitry Kurapeev, Maxim Popov, Marina Sidorova, Sarkis Minasian, Michael Galagudza, Anna Kostareva, Anton Fedorov
BACKGROUND: microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels...
June 2016: Biomolecular Detection and Quantification
Hua-Jun He, Jamie L Almeida, Steve P Lund, Carolyn R Steffen, Steve Choquette, Kenneth D Cole
NIST standard reference material (SRM) 2373 was developed to improve the measurements of the HER2 gene amplification in DNA samples. SRM 2373 consists of genomic DNA extracted from five breast cancer cell lines with different amounts of amplification of the HER2 gene. The five components are derived from the human cell lines SK-BR-3, MDA-MB-231, MDA-MB-361, MDA-MB-453, and BT-474. The certified values are the ratios of the HER2 gene copy numbers to the copy numbers of selected reference genes DCK, EIF5B, RPS27A, and PMM1...
June 2016: Biomolecular Detection and Quantification
S M Da Silva, L K Vang, N D Olson, S P Lund, A S Downey, Z Kelman, M L Salit, N J Lin, J B Morrow
AIMS: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). METHODS AND RESULTS: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration...
March 2016: Biomolecular Detection and Quantification
Oskar Karlsson, Lova Segerström, Robert Sjöback, Ingrid Nylander, Mats Borén
Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method...
March 2016: Biomolecular Detection and Quantification
Lars Gerdes, Azuka Iwobi, Ulrich Busch, Sven Pecoraro
Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample...
March 2016: Biomolecular Detection and Quantification
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