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Biomolecular Detection and Quantification

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https://www.readbyqxmd.com/read/27679763/science-in-the-uk-whereto-now
#1
EDITORIAL
Stephen Bustin
No abstract text is available yet for this article.
September 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27617230/validation-of-a-digital-pcr-method-for-quantification-of-dna-copy-number-concentrations-by-using-a-certified-reference-material
#2
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
https://www.readbyqxmd.com/read/27617229/influence-of-primer-probe-chemistry-and-amplification-target-on-reverse-transcription-digital-pcr-quantification-of-viral-rna
#3
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
https://www.readbyqxmd.com/read/27551672/methods-for-comparing-multiple-digital-pcr-experiments
#4
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
https://www.readbyqxmd.com/read/27551671/flexible-analysis-of-digital-pcr-experiments-using-generalized-linear-mixed-models
#5
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
https://www.readbyqxmd.com/read/27335808/diagnostic-ras-mutation-analysis-by-polymerase-chain-reaction-pcr
#6
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
https://www.readbyqxmd.com/read/27335807/an-international-comparability-study-on-quantification-of-mrna-gene-expression-ratios-ccqm-p103-1
#7
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
https://www.readbyqxmd.com/read/27335806/heparinase-treatment-of-heparin-contaminated-plasma-from-coronary-artery-bypass-grafting-patients-enables-reliable-quantification-of-micrornas
#8
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
https://www.readbyqxmd.com/read/27335805/development-of-nist-standard-reference-material-2373-genomic-dna-standards-for-her2-measurements
#9
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
https://www.readbyqxmd.com/read/27077050/evaluation-of-microbial-qpcr-workflows-using-engineered-saccharomyces-cerevisiae
#10
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
https://www.readbyqxmd.com/read/27077049/qpcr-based-mrna-quality-score-show-intact-mrna-after-heat-stabilization
#11
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
https://www.readbyqxmd.com/read/27077048/optimization-of-digital-droplet-polymerase-chain-reaction-for-quantification-of-genetically-modified-organisms
#12
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
https://www.readbyqxmd.com/read/27077047/improving-the-reliability-of-peer-reviewed-publications-we-are-all-in-it-together
#13
Stephen A Bustin, Tania Nolan
The current, and welcome, focus on standardization of techniques and transparency of reporting in the biomedical, peer-reviewed literature is commendable. However, that focus has been intermittent as well as lacklustre and so failed to tackle the alarming lack of reliability and reproducibly of biomedical research. Authors have access to numerous recommendations, ranging from simple standards dealing with technical issues to those regulating clinical trials, suggesting that improved reporting guidelines are not the solution...
March 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077046/real-time-pcr-probe-optimization-using-design-of-experiments-approach
#14
S Wadle, M Lehnert, S Rubenwolf, R Zengerle, F von Stetten
Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR)...
March 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077045/incidence-and-detection-of-beak-and-feather-disease-virus-in-psittacine-birds-in-the-uae
#15
F Hakimuddin, F Abidi, O Jafer, C Li, U Wernery, Ch Hebel, K Khazanehdari
Beak and feather disease is caused by Circovirus, which affects actively growing beak and feather cells of avian species. The disease affects mainly young birds while older birds may overcome the disease with few lasting effects. Due to lack of treatment, the only way to control the disease is through hygiene and early diagnosis. As a diagnostic tool, we have established a Taqman probe based real-time PCR assay to detect the presence of the viral genome in psittacine birds in UAE and reported the incidence of circovirus in different species of psittacine birds...
January 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077044/targeted-resequencing-and-variant-validation-using-pxlence-pcr-assays
#16
Frauke Coppieters, Kimberly Verniers, Kim De Leeneer, Jo Vandesompele, Steve Lefever
The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage...
January 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077043/evaluation-of-bias-associated-with-high-multiplex-target-specific-pre-amplification
#17
Steven T Okino, Michelle Kong, Haya Sarras, Yan Wang
We developed a novel PCR-based pre-amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million-fold. To assess potential bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis. We assessed three types of bias: amplification bias, dynamic range bias and fold-change bias. We show that our PreAmp workflow introduces only minimal amplification and fold-change bias under stringent conditions. We do detect dynamic range bias if a target gene is highly abundant and PreAmp occurred for 16 or more PCR cycles; however, this type of bias is easily correctable...
January 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077042/differential-amplicons-%C3%AE-amp-a-new-molecular-method-to-assess-rna-integrity
#18
J Björkman, D Švec, E Lott, M Kubista, R Sjöback
Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest...
January 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077041/pitfalls-in-pcr-troubleshooting-expect-the-unexpected
#19
Livia Schrick, Andreas Nitsche
PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings...
January 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27077040/feasibility-of-a-workflow-for-the-molecular-characterization-of-single-cells-by-next-generation-sequencing
#20
Francesca Salvianti, Giada Rotunno, Francesca Galardi, Francesca De Luca, Marta Pestrin, Alessandro Maria Vannucchi, Angelo Di Leo, Mario Pazzagli, Pamela Pinzani
The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer...
September 2015: Biomolecular Detection and Quantification
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