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Title: Exploration, quantification, and mitigation of systematic error in high-throughput approaches to gene-expression profiling : implications for data reproducibility
Author: Kitchen, Robert Raymond
ISNI:       0000 0004 2732 0549
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2011
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Technological and methodological advances in the fields of medical and life-sciences have, over the last 25 years, revolutionised the way in which cellular activity is measured at the molecular level. Three such advances have provided a means of accurately and rapidly quantifying mRNA, from the development of quantitative Polymerase Chain Reaction (qPCR), to DNA microarrays, and second-generation RNA-sequencing (RNA-seq). Despite consistent improvements in measurement precision and sample throughput, the data generated continue to be a ffected by high levels of variability due to the use of biologically distinct experimental subjects, practical restrictions necessitating the use of small sample sizes, and technical noise introduced during frequently complex sample preparation and analysis procedures. A series of experiments were performed during this project to pro le sources of technical noise in each of these three techniques, with the aim of using the information to produce more accurate and more reliable results. The mechanisms for the introduction of confounding noise in these experiments are highly unpredictable. The variance structure of a qPCR experiment, for example, depends on the particular tissue-type and gene under assessment while expression data obtained by microarray can be greatly influenced by the day on which each array was processed and scanned. RNA-seq, on the other hand, produces data that appear very consistent in terms of differences between technical replicates, however there exist large differences when results are compared against those reported by microarray, which require careful interpretation. It is demonstrated in this thesis that by quantifying some of the major sources of noise in an experiment and utilising compensation mechanisms, either pre- or post-hoc, researchers are better equipped to perform experiments that are more robust, more accurate, and more consistent.
Supervisor: van Hemert, Jano; Sims, Andy. ; Clarke, Peter. ; Ogilvie, Varrie. Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: gene-expression ; microarray ; qPCR ; quantitative Polymerase Chain Reaction ; RNA-seq ; technical noise ; batch effects ; reproducibility