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Technical variability is greater than biological variability in a microarray experiment but both are outweighed by changes induced by stimulation.

Bryant PA, Smyth GK, Robins-Browne R, Curtis N - PLoS ONE (2011)

Bottom Line: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated.Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components).Variability in gene expression was very low and likely to improve further as technical advances are made.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics, The University of Melbourne, Melbourne, Australia.

ABSTRACT

Introduction: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS).

Methods and results: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined.

Conclusions: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases.

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Related in: MedlinePlus

Variability of gene expression for each component level of the experiment.a) Standard deviation of all genes for each variance component, showing median standard deviation, interquartile ranges and outliers; b) Proportion of variance contributed by each component to the overall variance. Amp - amplification run.
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pone-0019556-g003: Variability of gene expression for each component level of the experiment.a) Standard deviation of all genes for each variance component, showing median standard deviation, interquartile ranges and outliers; b) Proportion of variance contributed by each component to the overall variance. Amp - amplification run.

Mentions: The median standard deviation (SD, σ) for all of the variance components was very small, at less than 0.1 for all components, and 0 for the majority (figure 3a). Note that ‘Residual’ refers to the variance component attributable to hybridisation, but also includes any other factors contributing to variability downstream of amplification. The greatest variability was at the levels of amplification and hybridisation, but even these were small. The proportion that each component contributed to the overall variance indicated that the greatest proportion of variance was contributed by Residual/Hybridisation, which was double that contributed by Amplification, and nearly four times that contributed by most of the remaining components (figure 3b). Although the variance between different days was slightly smaller than between different subjects, when the LPS interaction was taken into account, the interaction between Day and LPS contributed variability greater than the Subject or Subject:LPS interaction.


Technical variability is greater than biological variability in a microarray experiment but both are outweighed by changes induced by stimulation.

Bryant PA, Smyth GK, Robins-Browne R, Curtis N - PLoS ONE (2011)

Variability of gene expression for each component level of the experiment.a) Standard deviation of all genes for each variance component, showing median standard deviation, interquartile ranges and outliers; b) Proportion of variance contributed by each component to the overall variance. Amp - amplification run.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104982&req=5

pone-0019556-g003: Variability of gene expression for each component level of the experiment.a) Standard deviation of all genes for each variance component, showing median standard deviation, interquartile ranges and outliers; b) Proportion of variance contributed by each component to the overall variance. Amp - amplification run.
Mentions: The median standard deviation (SD, σ) for all of the variance components was very small, at less than 0.1 for all components, and 0 for the majority (figure 3a). Note that ‘Residual’ refers to the variance component attributable to hybridisation, but also includes any other factors contributing to variability downstream of amplification. The greatest variability was at the levels of amplification and hybridisation, but even these were small. The proportion that each component contributed to the overall variance indicated that the greatest proportion of variance was contributed by Residual/Hybridisation, which was double that contributed by Amplification, and nearly four times that contributed by most of the remaining components (figure 3b). Although the variance between different days was slightly smaller than between different subjects, when the LPS interaction was taken into account, the interaction between Day and LPS contributed variability greater than the Subject or Subject:LPS interaction.

Bottom Line: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated.Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components).Variability in gene expression was very low and likely to improve further as technical advances are made.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics, The University of Melbourne, Melbourne, Australia.

ABSTRACT

Introduction: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS).

Methods and results: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined.

Conclusions: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases.

Show MeSH
Related in: MedlinePlus