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Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)
Publication date: December 2017Source: Biomolecular Detection and Quantification, Volume 14Author(s): Adrián Ruiz-Villalba, Elizabeth van Pelt-Verkuil, Quinn D Gunst, Jan M Ruijter, Maurice JB van den HoffAbstractQuantitative 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 ...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

Next-generation sequencing applications in clinical bacteriology
Publication date: December 2017Source: Biomolecular Detection and Quantification, Volume 14Author(s): Yair Motro, Jacob Moran-GiladAbstractWith 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 (Source: Biomolecular Detecti...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

qPCR primer design revisited
We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers
Publication date: May 2018Source: Biomolecular Detection and Quantification, Volume 15Author(s): M.G.M. Kok, M.W.J. de Ronde, P.D. Moerland, J.M. Ruijter, E.E. Creemers, S.J. Pinto-SietsmaAbstractSince 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...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants
Publication date: May 2018Source: Biomolecular Detection and Quantification, Volume 15Author(s): S. Stasik, C. Schuster, C. Ortlepp, U. Platzbecker, M. Bornhäuser, J. Schetelig, G. Ehninger, G. Folprecht, C. ThiedeAbstractMonitoring 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 P...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples
Publication date: May 2018Source: Biomolecular Detection and Quantification, Volume 15Author(s): Bhaja K. Padhi, Manjeet Singh, Marianela Rosales, Guillaume Pelletier, Sabit CakmakAbstractReverse 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 expressi...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events
Publication date: May 2018Source: Biomolecular Detection and Quantification, Volume 15Author(s): Tigst Demeke, Monika EngAbstractDroplet 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 quantif...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

Improving the standardization of mRNA measurement by RT-qPCR
Publication date: May 2018Source: Biomolecular Detection and Quantification, Volume 15Author(s): Rebecca Sanders, Stephen Bustin, Jim Huggett, Deborah MasonAbstractHuman 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 experimen...
Source: Biomolecular Detection and Quantification - July 5, 2018 Category: Molecular Biology Source Type: research

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events
Publication date: May 2018 Source:Biomolecular Detection and Quantification, Volume 15 Author(s): 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 quantific...
Source: Biomolecular Detection and Quantification - April 10, 2018 Category: Molecular Biology Source Type: research

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples
Publication date: May 2018 Source:Biomolecular Detection and Quantification, Volume 15 Author(s): 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 express...
Source: Biomolecular Detection and Quantification - March 17, 2018 Category: Molecular Biology Source Type: research

Improving the standardization of mRNA measurement by RT-qPCR
Publication date: May 2018 Source:Biomolecular Detection and Quantification, Volume 15 Author(s): 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 experimen...
Source: Biomolecular Detection and Quantification - March 16, 2018 Category: Molecular Biology Source Type: research

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants
Publication date: May 2018 Source:Biomolecular Detection and Quantification, Volume 15 Author(s): 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 Torr...
Source: Biomolecular Detection and Quantification - January 9, 2018 Category: Molecular Biology Source Type: research

Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers
Publication date: May 2018 Source:Biomolecular Detection and Quantification, Volume 15 Author(s): 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 hi...
Source: Biomolecular Detection and Quantification - December 19, 2017 Category: Molecular Biology Source Type: research

qPCR primer design revisited
We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications. (Source: Biomolecular Detection and Quantification)
Source: Biomolecular Detection and Quantification - November 23, 2017 Category: Molecular Biology Source Type: research

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)
Publication date: December 2017 Source:Biomolecular Detection and Quantification, Volume 14 Author(s): 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...
Source: Biomolecular Detection and Quantification - November 1, 2017 Category: Molecular Biology Source Type: research