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Biomolecular Detection and Quantification

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https://www.readbyqxmd.com/read/29021971/evaluation-of-relative-quantification-of-alternatively-spliced-transcripts-using-droplet-digital-pcr
#1
Mattias Van Heetvelde, Wouter Van Loocke, Wim Trypsteen, Annelot Baert, Katrien Vanderheyden, Brecht Crombez, Jo Vandesompele, Kim De Leeneer, Kathleen B M Claes
INTRODUCTION: For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms...
September 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/29021970/quantification-of-mitochondrial-dna-copy-number-in-suspected-cancer-patients-by-a-well-optimized-ddpcr-method
#2
Ashfaque A Memon, Bengt Zöller, Anna Hedelius, Xiao Wang, Emelie Stenman, Jan Sundquist, Kristina Sundquist
Changes in mitochondrial DNA (mtDNA) content is a useful clinical biomarker for various diseases, however results are controversial as several analytical factors can affect measurement of mtDNA. MtDNA is often quantified by taking ratio between a target mitochondrial gene and a reference nuclear gene (mtDNA/nDNA) using quantitative real time PCR often on two separate experiments. It measures relative levels by using external calibrator which may not be comparable across laboratories. We have developed and optimized a droplet digital PCR (ddPCR) based method for quantification of absolute copy number of both mtDNA and nDNA gene in whole blood...
September 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/29021969/-k-means-and-cluster-models-for-cancer-signatures
#3
Zura Kakushadze, Willie Yu
We present *K-means clustering algorithm and source code by expanding statistical clustering methods applied in https://ssrn.com/abstract=2802753 to quantitative finance. *K-means is statistically deterministic without specifying initial centers, etc. We apply *K-means to extracting cancer signatures from genome data without using nonnegative matrix factorization (NMF). *K-means' computational cost is a fraction of NMF's. Using 1389 published samples for 14 cancer types, we find that 3 cancers (liver cancer, lung cancer and renal cell carcinoma) stand out and do not have cluster-like structures...
September 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/29021968/the-unyvero-p55-sample-in-answer-out-pneumonia-assay-a-performance-evaluation
#4
C Ozongwu, Y Personne, G Platt, C Jeanes, S Aydin, N Kozato, V Gant, J O'Grady, V I Enne
BACKGROUND: O'Neill's recent Review on Antimicrobial Resistance expressed the view that by 2020 high-income countries should make it mandatory to support antimicrobial prescribing with rapid diagnostic evidence whenever possible. METHODS: Routine microbiology diagnosis of 95 respiratory specimens from patients with severe infection were compared with those generated by the Unyvero P55 test, which detects 20 pathogens and 19 antimicrobial resistance markers. Supplementary molecular testing for antimicrobial resistance genes, comprehensive culture methodology and 16S rRNA sequencing were performed...
September 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28702368/how-to-speed-up-the-polymerase-chain-reaction
#5
Stephen A Bustin
Reducing the time taken to run qPCR assays on today's qPCR cyclers is rather straightforward and requires no specialised reagents or instruments. As the first article in a new series of short technical reports, I demonstrate that it is possible to reduce significantly both denaturation temperatures and cycling times, whilst retaining sensitivity and specificity of the original qPCR conditions.
June 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28702367/the-continuing-problem-of-poor-transparency-of-reporting-and-use-of-inappropriate-methods-for-rt-qpcr
#6
Stephen Bustin
Attendance at this year's European Calcified Tissue Society's (ECTS) Congress reveals that the methods used to obtain qPCR results continue to be significantly flawed and that and their reporting remain inadequate.
June 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28702366/methods-to-determine-limit-of-detection-and-limit-of-quantification-in-quantitative-real-time-pcr-qpcr
#7
Amin Forootan, Robert Sjöback, Jens Björkman, Björn Sjögreen, Lucas Linz, Mikael Kubista
Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR...
June 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28331816/inhibition-of-fat-cell-differentiation-in-3t3-l1-pre-adipocytes-by-all-trans-retinoic-acid-integrative-analysis-of-transcriptomic-and-phenotypic-data
#8
Katharina Stoecker, Steffen Sass, Fabian J Theis, Hans Hauner, Michael W Pfaffl
The process of adipogenesis is controlled in a highly orchestrated manner, including transcriptional and post-transcriptional events. In developing 3T3-L1 pre-adipocytes, this program can be interrupted by all-trans retinoic acid (ATRA). To examine this inhibiting impact by ATRA, we generated large-scale transcriptomic data on the microRNA and mRNA level. Non-coding RNAs such as microRNAs represent a field in RNA turnover, which is very important for understanding the regulation of mRNA gene expression. High throughput mRNA and microRNA expression profiling was performed using mRNA hybridisation microarray technology and multiplexed expression assay for microRNA quantification...
March 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28331815/measuring-e-coli-and-bacteriophage-dna-in-cell-sonicates-to-evaluate-the-cal1-reaction-as-a-synthetic-biology-standard-for-qpcr
#9
Alexander Templar, Desmond M Schofield, Darren N Nesbeth
We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the performance of the Linear Regression of Efficiency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biology standard. We firstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is amplified. Amplification efficiency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake flask cultivation...
March 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28331814/molecular-techniques-for-the-personalised-management-of-patients-with-chronic-myeloid-leukaemia
#10
REVIEW
Mary Alikian, Robert Peter Gale, Jane F Apperley, Letizia Foroni
Chronic myeloid leukemia (CML) is the paradigm for targeted cancer therapy. RT-qPCR is the gold standard for monitoring response to tyrosine kinase-inhibitor (TKI) therapy based on the reduction of blood or bone marrow BCR-ABL1. Some patients with CML and very low or undetectable levels of BCR-ABL1 transcripts can stop TKI-therapy without CML recurrence. However, about 60 percent of patients discontinuing TKI-therapy have rapid leukaemia recurrence. This has increased the need for more sensitive and specific techniques to measure residual CML cells...
March 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28331813/reproducibility-of-biomedical-research-the-importance-of-editorial-vigilance
#11
EDITORIAL
Stephen A Bustin, Jim F Huggett
Many journal editors are a failing to implement their own authors' instructions, resulting in the publication of many articles that do not meet basic standards of transparency, employ unsuitable data analysis methods and report overly optimistic conclusions. This problem is particularly acute where quantitative measurements are made and results in the publication of papers that lack scientific rigor and contributes to the concerns with regard to the reproducibility of biomedical research. This hampers research areas such as biomarker identification, as reproducing all but the most striking changes is challenging and translation to patient care rare...
March 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27990349/digital-polymerase-chain-reaction-for-characterisation-of-dna-reference-materials
#12
REVIEW
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
https://www.readbyqxmd.com/read/27990348/three-color-crystal-digital-pcr
#13
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
https://www.readbyqxmd.com/read/27990347/digital-pcr-dynamic-range-is-approaching-that-of-real-time-quantitative-pcr
#14
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
https://www.readbyqxmd.com/read/27990346/designing-and-interpretation-of-digital-assays-concentration-of-target-in-the-sample-and-in-the-source-of-sample
#15
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
https://www.readbyqxmd.com/read/27990345/fundamentals-of-multiplexing-with-digital-pcr
#16
REVIEW
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
https://www.readbyqxmd.com/read/27990344/applicability-of-digital-pcr-to-the-investigation-of-pediatric-onset-genetic-disorders
#17
REVIEW
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
https://www.readbyqxmd.com/read/27990343/homogeneous-and-digital-proximity-ligation-assays-for-the-detection-of-clostridium-difficile-toxins-a-and-b
#18
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
https://www.readbyqxmd.com/read/27990342/digital-pcr-a-technique-for-the-future
#19
EDITORIAL
Valerie Taly, Jim Huggett
No abstract text is available yet for this article.
December 2016: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/27679763/science-in-the-uk-whereto-now
#20
EDITORIAL
Stephen Bustin
No abstract text is available yet for this article.
September 2016: Biomolecular Detection and Quantification
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