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Design and Optimization of Reverse-Transcription Quantitative PCR Experiments
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SYSNO ASEP 0339069 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Design and Optimization of Reverse-Transcription Quantitative PCR Experiments Author(s) Tichopád, A. (DE)
Kitchen, R. (GB)
Riedmaier, I. (DE)
Becker, Ch. (DE)
Ståhlberg, A. (SE)
Kubista, Mikael (BTO-N) RIDSource Title Clinical Chemistry. - : Oxford University Press - ISSN 0009-9147
Roč. 55, č. 10 (2009), s. 1816-1823Number of pages 8 s. Language eng - English Country US - United States Keywords Design ; optimization ; RT qPCR Subject RIV EG - Zoology CEZ AV0Z50520701 - BTO-N (2007-2013) UT WOS 000270471300010 DOI 10.1373/clinchem.2009.126201 Annotation Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells.A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures,and single cells,and we recommend the use of RT replicates when working with blood Workplace Institute of Biotechnology Contact Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Year of Publishing 2010
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