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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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Matrix of scatterplots showing log2 ratios for replicate hybridisations at each level of the experiment.The fold changes for each sample are plotted against those for each of the other samples for gene expression after stimulation with LPS for two subjects: subject A (top right of diagonal) and subject B (bottom left). Comparisons are made at different levels of the experiment: hybridisation (Hyb2), amplification (Amp2), stimulation (Stim2), day (Day2) and subject (Subject2). Correlation coefficients between each pair of samples are shown in the bottom right of each box.
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pone-0019556-g002: Matrix of scatterplots showing log2 ratios for replicate hybridisations at each level of the experiment.The fold changes for each sample are plotted against those for each of the other samples for gene expression after stimulation with LPS for two subjects: subject A (top right of diagonal) and subject B (bottom left). Comparisons are made at different levels of the experiment: hybridisation (Hyb2), amplification (Amp2), stimulation (Stim2), day (Day2) and subject (Subject2). Correlation coefficients between each pair of samples are shown in the bottom right of each box.

Mentions: The hierarchy of levels of variability which underlie the experimental design are shown by comparing gene expression for the 2 subjects (A and B) who had replication at every level of the experiment (figure 2). Comparisons were made between gene expression (against the common reference) from one hybridisation and gene expression from hybridisations at each different level of replication. For replication at the subject level, one of the other subjects on the same day was randomly selected for comparison.


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)

Matrix of scatterplots showing log2 ratios for replicate hybridisations at each level of the experiment.The fold changes for each sample are plotted against those for each of the other samples for gene expression after stimulation with LPS for two subjects: subject A (top right of diagonal) and subject B (bottom left). Comparisons are made at different levels of the experiment: hybridisation (Hyb2), amplification (Amp2), stimulation (Stim2), day (Day2) and subject (Subject2). Correlation coefficients between each pair of samples are shown in the bottom right of each box.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019556-g002: Matrix of scatterplots showing log2 ratios for replicate hybridisations at each level of the experiment.The fold changes for each sample are plotted against those for each of the other samples for gene expression after stimulation with LPS for two subjects: subject A (top right of diagonal) and subject B (bottom left). Comparisons are made at different levels of the experiment: hybridisation (Hyb2), amplification (Amp2), stimulation (Stim2), day (Day2) and subject (Subject2). Correlation coefficients between each pair of samples are shown in the bottom right of each box.
Mentions: The hierarchy of levels of variability which underlie the experimental design are shown by comparing gene expression for the 2 subjects (A and B) who had replication at every level of the experiment (figure 2). Comparisons were made between gene expression (against the common reference) from one hybridisation and gene expression from hybridisations at each different level of replication. For replication at the subject level, one of the other subjects on the same day was randomly selected for comparison.

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