Statistical aspects of quantitative real-time PCR experiment design

0350829 - BTÚ 2011 RIV US eng J - Journal Article
Kitchen, R.R. - Kubista, Mikael - Tichopád, Aleš
Statistical aspects of quantitative real-time PCR experiment design.
Methods. Roč. 50, č. 4 (2010), s. 231-236. ISSN 1046-2023. E-ISSN 1095-9130
R&D Projects: GA AV ČR IAA500520809
Institutional research plan: CEZ:AV0Z50520701
Keywords : Real-time PCR * Experiment design * Nested analysis of variance
Subject RIV: EB - Genetics ; Molecular Biology
Impact factor: 4.527, year: 2010

Experiments using qPCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of variability through optimum use of biological and technical replicates. Calculations rely on knowledge of the expected variances of the measurements of each group of samples and the magnitude of the treatment effect. Here we introduce a method that exploits a small pilot study to estimate the biological and technical variances in order to improve the design of a subsequent large experiment. We measure the variance contributions at several ‘levels’ of the experiment design and provide a means of using this information to predict both the total variance and the prospective power of the assay. We also discuss the effect of normalisation to a reference gene in terms of the measured variance components of the gene of interest. Finally, we describe a software implementation of these methods, powerNest
Permanent Link: http://hdl.handle.net/11104/0190725