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1دورية أكاديمية
المؤلفون: Stephen Bustin, Claire Bustin, Sara Kirvell, Tania Nolan, Reinhold D. Mueller, Gregory L. Shipley
مصطلحات موضوعية: PCR, qPCR, sensitivity, PCR efficiency, Molecular diagnostics
الوصف: The COVID-19 pandemic resulted in a universal, immediate, and vast demand for comprehensive molecular diagnostic testing, especially real-time quantitative (qPCR)-based methods. This rapidly triggered a global shortage of testing capacity, equipment, and reagents. Even today, supply times for chemicals from date of order to delivery are often much longer than pre-pandemic. Furthermore, many companies have ratcheted up the price for minimum volumes of reaction master mixes essential for qPCR assays, causing additional problems for academic laboratories often operating on a shoestring. We have validated two strategies that stretch reagent supplies and, whilst particularly applicable in case of scarcity, can readily be incorporated into standard qPCR protocols, with appropriate validation. The first strategy demonstrates equivalent performance of a selection of “past expiry date” and newly purchased master mixes. This approach is valid for both standard and fast qPCR protocols. The second validates the use of these master mixes at less than 1x final concentration without loss of qPCR efficiency or sensitivity.
العلاقة: 10779/aru.23782362.v1; https://figshare.com/articles/journal_contribution/Maximising_the_Use_of_Scarce_qPCR_Master_Mixes/23782362Test
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2دورية أكاديمية
مصطلحات موضوعية: COVID-19, SARS-CoV-2, molecular diagnostics, quantification cycle, RT-qPCR
الوصف: Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.
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3دورية أكاديمية
المؤلفون: Jim F. Huggett, Alexandra S. Whale, Ward De Spiegelaere, Wim Trypsteen, Afif Abdel Nour, Young-Kyung Bae, Vladimir Benes, Daniel Burke, Megan Cleveland, Philippe Corbisier, Alison S. Devonshire, Lianhua Dong, Daniela Drandi, Carole A. Foy, Jeremy A. Garson, Hua-Jun He, Jan Hellemans, Mikael Kubista, Antoon Lievens, Mike G. Makrigiorgos, Mojca Milavec, Reinhold D. Mueller, Tania Nolan, Denise M. O’Sullivan, Michael W. Pfaffl, Stefan Rödiger, Erica L. Romsos, Gregory L. Shipley, Valerie Taly, Andreas Untergasser, Carl T. Wittwer, Stephen A. Bustin, Jo Vandesompele
مصطلحات موضوعية: research
الوصف: Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.
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4دورية أكاديمية
المؤلفون: Jim F. Huggett, Carole A. Foy, Vladimir Benes, Kerry Emslie, Jeremy A. Garson, Ross Haynes, Jan Hellemans, Mikael Kubista, Reinhold D. Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, Carl T. Wittwer, Stephen A. Bustin
مصطلحات موضوعية: research
الوصف: There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.
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5دورية أكاديمية
المؤلفون: Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim F. Huggett, Mikael Kubista, Reinhold D. Mueller, Tania Nolan, Michael W. Pfaffl, Gregory Shipley, Carl T. Wittwer, Peter Schjerling, Philip J. Day, Mónica Abreu, Begoña Aguado, Jean-François Beaulieu, Anneleen Beckers, Sara Bogaert, John A. Browne, Fernando Carrasco-Ramiro, Liesbeth Ceelen, Kate Ciborowski, Pieter Cornillie, Stephanie Coulon, Ann Cuypers, Sara De Brouwer, Leentje De Ceuninck, Jurgen De Craene, Hélène De Naeyer, Ward De Spiegelaere, Kato Deckers, Annelies Dheedene, Kaat Durinck, Margarida Ferreira-Teixeira, Annelies Fieuw, Jack M. Gallup, Sandra Gonzalo-Flores, Karen Goossens, Femke Heindryckx, Elizabeth Herring, Hans Hoenicka, Laura Icardi, Rolf Jaggi, Farzad Javad, Michael Karampelias, Frederick Kibenge, Molly Kibenge, Candy Kumps, Irina Lambertz, Tim Lammens, Amelia Markey, Peter Messiaen, Evelien Mets, Sofia Morais, Alberto Mudarra-Rubio, Justine Nakiwala, Hilde Nelis, Pal A. Olsvik, Claudina Pérez-Novo, Michelle Plusquin, Tony Remans, Ali Rihani, Paulo Rodrigues-Santos, Pieter Rondou, Rebecca Sanders, Katharina Schmidt-Bleek, Kerstin Skovgaard, Karen Smeets, Laura Tabera, Stefan Toegel, Tim Van Acker, Wim Van den Broeck, Joni Van der Meulen, Mireille Van Gele, Gert Van Peer, Mario Van Poucke, Nadine Van Roy, Sarah Vergult, Joris Wauman, Marina Tshuikina-Wiklander, Erik Willems, Sara Zaccara, Fjoralba Zeka, Jo Vandesompele
مصطلحات موضوعية: research
الوصف: Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
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6
المؤلفون: Patrick C. Hallenbeck, Reinhold D. Mueller
المصدر: Analytical Biochemistry. 138:189-195
مصطلحات موضوعية: Chromatography, biology, Physarum, Abundance (chemistry), Biophysics, Analytical chemistry, Aqueous two-phase system, Physarum polycephalum, Cell Biology, biology.organism_classification, Biochemistry, High-performance liquid chromatography, Histones, chemistry.chemical_compound, Histone, chemistry, Phase (matter), biology.protein, Electrophoresis, Polyacrylamide Gel, Acetonitrile, Molecular Biology, Chromatography, High Pressure Liquid
الوصف: The histones of Physarum polycephalum were efficiently separated using a muBondapak C-18 column with 50 mM triethylamine-phosphoric acid, pH 2.2, as the aqueous phase and a gradient of increasing concentration of acetonitrile. The effects of buffer concentration and changes in gradient rate and flow rate on retention times were investigated to optimize separation. The recovery of total histones (70%) was independent of loading up to at least 0.5 mg. Loading had little effect on retention times. Under optimal conditions, substantially pure (less than 90%) fractions of each histone were obtained. The quantity of each histone after HPLC fractionation reflected its abundance in the original sample, indicating that there was no selective loss of a particular histone.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::23ba821577634f3ae033a1100e48578eTest
https://doi.org/10.1016/0003-2697Test(84)90787-5 -
7دورية أكاديمية
المؤلفون: Huggett, J. F., Whale, A. S., Spiegelaere, W. De, Trypsteen, W., Nour, A. A., Bae, Y.-K., Benes, V., Burke, D. G., Corbisier, Philippe, Cleveland, Megan, Devonshire, Alison S, Dong, Lianhua, Drandi, Deniela, Foy, Carole, Garson, J. A., Hellemans, J., He, H.-J., Kubista, M., Lievens, A., Makrigiorgos, M., Milavec, M., Mueller, R. D., Nolan, T., O'Sullivan, D., Pfaffl, M. W., Roediger, S., Romsos, E., Shipley, G., Taly, V., Untergasser, A., Wittwer, C., Bustin, S., Vandesompele., And J.
المساهمون: University of Surrey (UNIS), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris Sciences et Lettres (PSL)-Université Paris Sciences et Lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), dMIQE Group: Alexandra S Whale, Ward De Spiegelaere, Wim Trypsteen, Afif Abdel Nour, Young-Kyung Bae, Vladimir Benes, Daniel Burke, Megan Cleveland, Philippe Corbisier, Alison S Devonshire, Lianhua Dong, Daniela Drandi, Carole A Foy, Jeremy A Garson, Hua-Jun He, Jan Hellemans, Mikael Kubista, Antoon Lievens, Mike G Makrigiorgos, Mojca Milavec, Reinhold D Mueller, Tania Nolan, Denise M O'Sullivan, Michael W Pfaffl, Stefan Rödiger, Erica L Romsos, Gregory L Shipley, Valerie Taly, Andreas Untergasser, Carl T Wittwer, Stephen A Bustin, Jo Vandesompele
المصدر: ISSN: 0009-9147.
مصطلحات موضوعية: MIQE, dMIQE, dPCR, digital PCR, [SDV]Life Sciences [q-bio]
الوصف: International audience ; Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.
العلاقة: info:eu-repo/semantics/altIdentifier/pmid/32746458; inserm-02992959; https://inserm.hal.science/inserm-02992959Test; https://inserm.hal.science/inserm-02992959/documentTest; https://inserm.hal.science/inserm-02992959/file/hvaa125.pdfTest; PUBMED: 32746458
الإتاحة: https://doi.org/10.1093/clinchem/hvaa125Test
https://inserm.hal.science/inserm-02992959Test
https://inserm.hal.science/inserm-02992959/documentTest
https://inserm.hal.science/inserm-02992959/file/hvaa125.pdfTest -
8دورية أكاديمية
المؤلفون: Popodi, Ellen M.
المساهمون: Gail L. Waring, A. Krishna Kumaran, Reinhold D. Mueller
المصدر: Dissertations (1934 -)
مصطلحات موضوعية: Biology
الوصف: The formation of the insect eggshell is a popular model system in which to study molecular mechanisms involved in the production of complex structures. The eggshell, secreted by ovarian follicle cells during the later stages of oocyte maturation, is deposited in several layers. The vitelline membrane is deposited during stages 8-10, and the several layers of the chorion are secreted during stages 11-14. We are examining the expression of early eggshell genes. I have characterized a cluster of follicle cell specific genes expressed during the period of vitelline membrane formation in Drosophila. Four genes are clustered in 8 kilobase pairs of genomic DNA from the 26A region of the second chromosome. Three of these genes encode 700 nucleotide long RNAs; the two most abundantly expressed code for major vitelline membrane proteins. Hybrid selected translation and sequencing studies suggest the product of the third gene is a minor eggshell protein. The fourth gene of the cluster specifies a 1400 nucleotide RNA of unknown function. The direction of transcription, intron-less structures and positions of these genes have been determined by northern blot analysis and S1 protection studies. Northern blot analysis demonstrates that these genes are expressed specifically in the female's ovary and are only detected in eggchambers of stages producing vitelline membrane. The follicle cell specific expression of these genes has been demonstrated by in situ hybridization to ovarian tissue sections or by S1 protection studies with RNA isolated from follicle cell or nurse cell fractions of cut stage 10 eggchambers. Gel electrophoresis-DNA binding studies with nuclear proteins isolated from expressing tissue have identified two potential cis-regulatory regions of one of the major vitelline membrane protein genes. One region is 5$\sp\prime$ to the gene, between $-$146 and $-$53; the second region is within the transcribed portion of the gene, upstream of +153. The location of the 5$\sp\prime$ region is similar to that of elements ...
وصف الملف: application/pdf
العلاقة: https://epublications.marquette.edu/dissertations_mu/3009Test; https://epublications.marquette.edu/context/dissertations_mu/article/4001/viewcontent/Popod_E_1989.pdfTest
