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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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Experimental design showing samples used to compare variability.Samples: Subject A–E, Day 1 and 2, LPS-stimulated sample (Stim) 1 and 2, amplification run (Amp) 1–3, and hybridisation (Hyb) 1–3. For each subject on each day there was also an unstimulated (Unstim) sample at 0 hours.
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pone-0019556-g001: Experimental design showing samples used to compare variability.Samples: Subject A–E, Day 1 and 2, LPS-stimulated sample (Stim) 1 and 2, amplification run (Amp) 1–3, and hybridisation (Hyb) 1–3. For each subject on each day there was also an unstimulated (Unstim) sample at 0 hours.

Mentions: An in vitro stimulation experiment was undertaken with replication at 5 levels (subject, day, stimulation tube, amplification and hybridisation) (figure 1). Five subjects each had blood taken on 2 days and their peripheral blood mononuclear cells (PBMC) were separated and stimulated with LPS in 2 parallel tubes for 24 hours or left unstimulated. All samples were amplified and RNA was hybridised against common reference RNA from pooled unstimulated samples from all subjects. Replication was also included at the amplification and hybridisation steps. Differences in gene expression with LPS stimulation were measured by comparing the log2 ratio of the stimulated samples with the unstimulated samples. Variability in gene expression was compared between all samples at baseline and after LPS stimulation.


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)

Experimental design showing samples used to compare variability.Samples: Subject A–E, Day 1 and 2, LPS-stimulated sample (Stim) 1 and 2, amplification run (Amp) 1–3, and hybridisation (Hyb) 1–3. For each subject on each day there was also an unstimulated (Unstim) sample at 0 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019556-g001: Experimental design showing samples used to compare variability.Samples: Subject A–E, Day 1 and 2, LPS-stimulated sample (Stim) 1 and 2, amplification run (Amp) 1–3, and hybridisation (Hyb) 1–3. For each subject on each day there was also an unstimulated (Unstim) sample at 0 hours.
Mentions: An in vitro stimulation experiment was undertaken with replication at 5 levels (subject, day, stimulation tube, amplification and hybridisation) (figure 1). Five subjects each had blood taken on 2 days and their peripheral blood mononuclear cells (PBMC) were separated and stimulated with LPS in 2 parallel tubes for 24 hours or left unstimulated. All samples were amplified and RNA was hybridised against common reference RNA from pooled unstimulated samples from all subjects. Replication was also included at the amplification and hybridisation steps. Differences in gene expression with LPS stimulation were measured by comparing the log2 ratio of the stimulated samples with the unstimulated samples. Variability in gene expression was compared between all samples at baseline and after LPS stimulation.

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