Statistical aspects of quantitative real-time PCR experiment design

Methods : a Companion to Methods in Enzymology
Robert R KitchenAles Tichopad

Abstract

Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on knowledge of the expected variances of the measurements of each group of samples and the magnitude of the treatment effect; the estimation of which is often uninformed and unreliable. 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. A validation of the method is provided through a variance analysis of representative genes in several bovine tissue-types. 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, powerNe...Continue Reading

References

Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
Feb 8, 2006·Molecular Aspects of Medicine·Mikael KubistaNeven Zoric
Jul 18, 2008·BMC Molecular Biology·Martin BengtssonAnders Ståhlberg

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Citations

Jan 1, 2014·Genome Medicine·Carlos Stahlhut, Frank J Slack
Apr 15, 2016·Biomolecular Detection and Quantification·S WadleF von Stetten
Mar 26, 2016·Frontiers in Plant Science·Jose V DieLisa J Rowland
Feb 24, 2016·ELife·Gilad D EvronyChristopher A Walsh
May 9, 2012·Wiley Interdisciplinary Reviews. RNA·Pauline Chugh, Dirk P Dittmer
Jun 9, 2016·Expert Review of Molecular Diagnostics·Nilanjana GhoshKoel Chaudhury
Aug 7, 2017·Journal of Biomedical Materials Research. Part a·Annika JuhlinMargarita Trobos
Jun 16, 2018·Clinical Chemistry·Maurice W J de RondeSara-Joan Pinto-Sietsma
Mar 15, 2013·Current Protocols in Chemical Biology·Joshua A Bittker
Aug 9, 2020·Frontiers in Cellular and Infection Microbiology·Jessica A GrembiAlfred M Spormann
Jan 9, 2021·Journal of Veterinary Internal Medicine·Lucia UngerEman Hamza
May 1, 2021·Synthetic Biology·Nicholas FacklerMichael Köpke
Jun 3, 2021·Life·Adrián Ruiz-VillalbaMaurice J B van den Hoff

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