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Biomolecular Detection and Quantification

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https://read.qxmd.com/read/30984566/development-of-event-specific-qpcr-detection-methods-for-genetically-modified-alfalfa-events-j101-j163-and-kk179
#1
Patrick Guertler, Lutz Grohmann, Heike Naumann, Melanie Pavlovic, Ulrich Busch
Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30956949/editorial
#2
EDITORIAL
Michael W Pfaffl
No abstract text is available yet for this article.
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30923679/the-emerging-role-of-cell-free-dna-as-a-molecular-marker-for-cancer-management
#3
REVIEW
Abel Jacobus Bronkhorst, Vida Ungerer, Stefan Holdenrieder
An increasing number of studies demonstrate the potential use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. However, harnessing the full potential of cfDNA requires (i) the optimization and standardization of preanalytical steps, (ii) refinement of current analysis strategies, and, perhaps most importantly, (iii) significant improvements in our understanding of its origin, physical properties, and dynamics in circulation. The latter knowledge is crucial for interpreting the associations between changes in the baseline characteristics of cfDNA and the clinical manifestations of cancer...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30923678/investigation-of-direct-counting-and-sizing-of-dna-fragments-in-flow-applying-an-improved-data-analysis-and-correction-method
#4
Martin Hussels, Susanne Engel, Nicole Bock
Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30923677/next-generation-sequencing-of-hiv-1-single-genome-amplicons
#5
Gustavo H Kijak, Eric Sanders-Buell, Phuc Pham, Elizabeth A Harbolick, Celina Oropeza, Anne Marie O'Sullivan, Meera Bose, Charmagne G Beckett, Mark Milazzo, Merlin L Robb, Sheila A Peel, Paul T Scott, Nelson L Michael, Adam W Armstrong, Jerome H Kim, David M Brett-Major, Sodsai Tovanabutra
The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30906693/considerations-and-quality-controls-when-analyzing-cell-free-tumor-dna
#6
Gustav Johansson, Daniel Andersson, Stefan Filges, Junrui Li, Andreas Muth, Tony E Godfrey, Anders Ståhlberg
Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30886826/challenging-the-proposed-causes-of-the-pcr-plateau-phase
#7
Linda Jansson, Johannes Hedman
Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30886825/corrigendum-to-incidence-and-detection-of-beak-and-feather-disease-virus-in-psittacine-birds-in-the-uae-biomol-detect-quantif-6-january-2016-27-32
#8
F Hakimuddin, F Abidi, O Jafer, C Li, U Wernery, Ch Hebel, K Khazanehdari
[This corrects the article DOI: 10.1016/j.bdq.2015.10.001.].
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30805297/shedding-light-the-importance-of-reverse-transcription-efficiency-standards-in-data-interpretation
#9
Jessica Schwaber, Stacey Andersen, Lars Nielsen
The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ability to do quantitative transcriptomics analyses. Multiple studies have focused on ways to optimise this process, resulting in contradictory recommendations. Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method's impact on subsequent data analysis. Using synthetic RNA standards, an example experiment is presented, outlining a method to (1) determine relevant efficiency and variability values and then to (2) incorporate this information into downstream analyses as a way to improve the accuracy of quantitative transcriptomics experiments...
March 2019: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30560063/essential-role-of-polymerases-for-assay-performance-impact-of-polymerase-replacement-in-a-well-established-assay
#10
Anna Kristina Witte, Romana Sickha, Patrick Mester, Susanne Fister, Dagmar Schoder, Peter Rossmanith
The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase...
December 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30560062/using-ddpcr-to-assess-the-dna-yield-of-ffpe-samples
#11
X J David Lu, Kelly Y P Liu, Yuqi Sarah Zhu, Cindy Cui, Catherine F Poh
Objectives: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples...
December 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/30560061/algorithms-for-automated-detection-of-hook-effect-bearing-amplification-curves
#12
Michał Burdukiewicz, Andrej-Nikolai Spiess, Konstantin A Blagodatskikh, Werner Lehmann, Peter Schierack, Stefan Rödiger
Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect ...
December 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29922591/effect-of-endogenous-reference-genes-on-digital-pcr-assessment-of-genetically-engineered-canola-events
#13
Tigst Demeke, Monika Eng
Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR...
May 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29922590/a-pcr-based-quantitative-assay-for-the-evaluation-of-mrna-integrity-in-rat-samples
#14
Bhaja K Padhi, Manjeet Singh, Marianela Rosales, Guillaume Pelletier, Sabit Cakmak
Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies...
May 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29922589/improving-the-standardization-of-mrna-measurement-by-rt-qpcr
#15
Rebecca Sanders, Stephen Bustin, Jim Huggett, Deborah Mason
Human health and safety depend on reliable measurements in medical diagnosis and on tests that support the selection and evaluation of therapeutic intervention and newly discovered molecular biomarkers must pass a rigorous evaluation process if they are to be of benefit to patients. Measurement standardization helps to maximize data quality and confidence and ultimately improves the reproducibility of published research. Failure to consider how a given experiment may be standardized can be costly, both financially as well as in time and failure to perform and report pre-clinical research in an appropriately rigorous manner will hinder the development of diagnostic methods...
May 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29349042/an-optimized-targeted-next-generation-sequencing-approach-for-sensitive-detection-of-single-nucleotide-variants
#16
S Stasik, C Schuster, C Ortlepp, U Platzbecker, M Bornhäuser, J Schetelig, G Ehninger, G Folprecht, C Thiede
Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. As a model case we used the c.1849G > T (p.Val617Phe) mutation of the JAK2 -gene. Several reaction parameters (e...
May 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29276692/small-sample-sizes-in-high-throughput-mirna-screens-a-common-pitfall-for-the-identification-of-mirna-biomarkers
#17
M G M Kok, M W J de Ronde, P D Moerland, J M Ruijter, E E Creemers, S J Pinto-Sietsma
Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. In order to identify circulating miRNA candidate biomarkers, in general, first an unbiased high-throughput screen is performed in which a large number of miRNAs is detected and quantified in the circulation...
May 2018: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29255685/amplification-of-nonspecific-products-in-quantitative-polymerase-chain-reactions-qpcr
#18
Adrián Ruiz-Villalba, Elizabeth van Pelt-Verkuil, Quinn D Gunst, Jan M Ruijter, Maurice Jb van den Hoff
Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input...
December 2017: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29255684/next-generation-sequencing-applications-in-clinical-bacteriology
#19
REVIEW
Yair Motro, Jacob Moran-Gilad
With the rapid advances in next generation sequencing (NGS) technologies, clinical and public health microbiology laboratories are increasingly adopting NGS technology in their workflows into their existing diagnostic cycles. In this bacteriology focused review, we review aspects and considerations for applying NGS in the clinical microbiology settings, and highlight the impact of such implementation on the analytical and post-analytical stages of diagnosis.
December 2017: Biomolecular Detection and Quantification
https://read.qxmd.com/read/29201647/qpcr-primer-design-revisited
#20
REVIEW
Stephen Bustin, Jim Huggett
Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range...
December 2017: Biomolecular Detection and Quantification
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