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Status: Bibliographieeintrag

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Verfasst von:Untergasser, Andreas [VerfasserIn]   i
 Ruijter, Jan M. [VerfasserIn]   i
 Benes, Vladimir [VerfasserIn]   i
 van den Hoff, Maurice J. B. [VerfasserIn]   i
Titel:Web-based LinRegPCR
Titelzusatz:application for the visualization and analysis of (RT)-qPCR amplification and melting data
Verf.angabe:Andreas Untergasser, Jan M. Ruijter, Vladimir Benes and Maurice J.B. van den Hoff
E-Jahr:2021
Jahr:24 August 2021
Umfang:18 S.
Fussnoten:Gesehen am 13.10.2021
Titel Quelle:Enthalten in: BMC bioinformatics
Ort Quelle:London : BioMed Central, 2000
Jahr Quelle:2021
Band/Heft Quelle:22(2021), Artikel-ID 398, Seite 1-18
ISSN Quelle:1471-2105
Abstract:Background The analyses of amplification and melting curves have been shown to provide valuable information on the quality of the individual reactions in quantitative PCR (qPCR) experiments and to result in more reliable and reproducible quantitative results. Implementation The main steps in the amplification curve analysis are (1) a unique baseline subtraction, not using the ground phase cycles, (2) PCR efficiency determination from the exponential phase of the individual reactions, (3) setting a common quantification threshold and (4) calculation of the efficiency-corrected target quantity with the common threshold, efficiency per assay and C-q per reaction. The melting curve analysis encompasses smoothing of the observed fluorescence data, normalization to remove product-independent fluorescence loss, peak calling and assessment of the correct peak by comparing its melting temperature with the known melting temperature of the intended amplification product. Results The LinRegPCR web application provides visualization and analysis of a single qPCR run. The user interface displays the analysis results on the amplification curve analysis and melting curve analysis in tables and graphs in which deviant reactions are highlighted. The annotated results in the tables can be exported for calculation of gene-expression ratios, fold-change between experimental conditions and further statistical analysis. Web-based LinRegPCR addresses two types of users, wet-lab scientists analyzing the amplification and melting curves of their own qPCR experiments and bioinformaticians creating pipelines for analysis of series of qPCR experiments by splitting its functionality into a stand-alone back-end RDML (Real-time PCR Data Markup Language) Python library and several companion applications for data visualization, analysis and interactive access. The use of the RDML data standard enables machine independent storage and exchange of qPCR data and the RDML-Tools assist with the import of qPCR data from the files exported by the qPCR instrument. Conclusions The combined implementation of these analyses in the newly developed web-based LinRegPCR () is platform independent and much faster than the original Windows-based versions of the LinRegPCR program. Moreover, web-based LinRegPCR includes a novel statistical outlier detection and the combination of amplification and melting curve analyses allows direct validation of the amplification product and reporting of reactions that amplify artefacts.
DOI:doi:10.1186/s12859-021-04306-1
URL:Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.

Volltext ; Verlag: https://doi.org/10.1186/s12859-021-04306-1
 Volltext: https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=DynamicDOIArticle&SrcApp=WOS&KeyAID=10.1186%2 ...
 DOI: https://doi.org/10.1186/s12859-021-04306-1
Datenträger:Online-Ressource
Sprache:eng
Sach-SW:Amplification curve
 guidelines
 LinRegPCR
 Melting curve
 pcr
 qPCR
 quantification
 rdml
 resolution
K10plus-PPN:1773559974
Verknüpfungen:→ Zeitschrift

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