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Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing
Publication date: Available online 12 September 2015 Source:Biomolecular Detection and Quantification Author(s): 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 ...
Source: Biomolecular Detection and Quantification - September 13, 2015 Category: Molecular Biology Source Type: research

Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing
Conclusion In targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of confidence limits and limit of detection for copy number measurement, and of frequency for each actionable mutation. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - August 29, 2015 Category: Molecular Biology Source Type: research

Effects of post-mortem and physical degradation on RNA integrity and quality
Publication date: Available online 21 August 2015 Source:Biomolecular Detection and Quantification Author(s): Monika Sidova, Silvie Tomankova, Pavel Abaffy, Mikael Kubista, Radek Sindelka The precision and reliability of quantitative nucleic acid analysis depends on the quality of the sample analyzed and the integrity of the nucleic acids. The integrity of RNA is currently primarily assessed by the analysis of ribosomal RNA, which is the by far dominant species. The extrapolation of these results to mRNAs and microRNAs, which are structurally quite different, is questionable. Here we show that ribosomal and s...
Source: Biomolecular Detection and Quantification - August 22, 2015 Category: Molecular Biology Source Type: research

Removal of between-run variation in a multi-plate qPCR experiment
Publication date: Available online 30 July 2015 Source:Biomolecular Detection and Quantification Author(s): Jan M. Ruijter, Adrián Ruiz Villalba, Jan Hellemans, Andreas Untergasser, Maurice J.B. van den Hoff Quantitative PCR (qPCR) is the method of choice in gene expression analysis. However, the number of groups or treatments, target genes and technical replicates quickly exceeds the capacity of a single run on a qPCR machine and the measurements have to be spread over more than 1 plate. Such multi-plate measurements often show similar proportional differences between experimental conditions, but different ...
Source: Biomolecular Detection and Quantification - July 31, 2015 Category: Molecular Biology Source Type: research

Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery
In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - June 8, 2015 Category: Molecular Biology Source Type: research

A glance at the applications of Singular Spectrum Analysis in gene expression data
Publication date: June 2015 Source:Biomolecular Detection and Quantification, Volume 4 Author(s): Hossein Hassani , Zara Ghodsi In recent years Singular Spectrum Analysis (SSA) has been used to solve many biomedical issues and is currently accepted as a potential technique in quantitative genetics studies. Presented in this article is a review of recent published genetics studies which have taken advantage of SSA. Since Singular Value Decomposition (SVD) is an important stage of this technique which can also be used as an independent analytical method in gene expression data, we also briefly touch upon some areas of ...
Source: Biomolecular Detection and Quantification - May 30, 2015 Category: Molecular Biology Source Type: research

Proximity assays for sensitive quantification of proteins
Publication date: June 2015 Source:Biomolecular Detection and Quantification, Volume 4 Author(s): Christina Greenwood , David Ruff , Sara Kirvell , Gemma Johnson , Harvinder S. Dhillon , Stephen A. Bustin Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can...
Source: Biomolecular Detection and Quantification - May 20, 2015 Category: Molecular Biology Source Type: research

Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study
Publication date: June 2015 Source:Biomolecular Detection and Quantification, Volume 4 Author(s): Vicente Sancenon , Wei Hau Goh , Aishwarya Sundaram , Kai Shih Er , Nidhi Johal , Svetlana Mukhina , Grant Carr , Saravanakumar Dhakshinamoorthy The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the d...
Source: Biomolecular Detection and Quantification - April 28, 2015 Category: Molecular Biology Source Type: research

Assessing the performance of the Oxford Nanopore Technologies MinION
In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G+C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2kb and 1kb respectively, while the longest single read was 98kb. The whole length of a 5kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate li...
Source: Biomolecular Detection and Quantification - March 24, 2015 Category: Molecular Biology Source Type: research

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments
Publication date: Available online 11 March 2015 Source:Biomolecular Detection and Quantification Author(s): David Svec , Ales Tichopad , Vendula Novosadova , Michael W. Pfaffl , Mikael Kubista We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qP...
Source: Biomolecular Detection and Quantification - March 11, 2015 Category: Molecular Biology Source Type: research

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons
This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11...
Source: Biomolecular Detection and Quantification - March 5, 2015 Category: Molecular Biology Source Type: research

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges
Conclusion The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - January 22, 2015 Category: Molecular Biology Source Type: research

The reproducibility of biomedical research: Sleepers awake!
This article argues that an important reason for this is the inappropriate use of molecular techniques, particularly in the field of RNA biomarkers, coupled with a tendency to exaggerate the importance of research findings. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - January 22, 2015 Category: Molecular Biology Source Type: research

Characterization of non classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges
Conclusion The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - January 20, 2015 Category: Molecular Biology Source Type: research

qPCR, dPCR, NGS – a journey
Publication date: Available online 15 January 2015 Source:Biomolecular Detection and Quantification Author(s): Jim Huggett , Justin O’Grady , Stephen Bustin (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - January 16, 2015 Category: Molecular Biology Source Type: research