Design and Optimization of Reverse-Transcription Quantitative PCR Experiments

0339069 - BTÚ 2010 RIV US eng J - Journal Article
Tichopád, A. - Kitchen, R. - Riedmaier, I. - Becker, Ch. - Ståhlberg, A. - Kubista, Mikael
Design and Optimization of Reverse-Transcription Quantitative PCR Experiments.
Clinical Chemistry. Roč. 55, č. 10 (2009), s. 1816-1823. ISSN 0009-9147. E-ISSN 1530-8561
Institutional research plan: CEZ:AV0Z50520701
Keywords : Design * optimization * RT qPCR
Subject RIV: EG - Zoology
Impact factor: 6.263, year: 2009

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
Permanent Link: http://hdl.handle.net/11104/0182679