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Technical analysis of cDNA microarrays.

Scott CP, VanWye J, McDonald MD, Crawford DL - PLoS ONE (2009)

Bottom Line: Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization.Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression.However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida, United States of America.

ABSTRACT

Background: There is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization.

Methodology/principal findings: In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course.

Conclusion: There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise.

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Related in: MedlinePlus

Gene expression for Single Blood isolate.Heat map for single blood isolate that was divided into four. RNA was purified, labeled and hybridized separately for each sample. Red is greater and green is less than the average gene specific fluorescence. First column (P) is the p-value from a one-way ANOVA. Only 6 genes (2.3%) out of 252 are significant at a critical p-value of 0.01. P-values (−log10) shown in the heat map are from an ANOVA for significant differences among samples using the 8 replicates for each separate RNA isolation. Color bar gives fold difference for log2 gene expression (e.g., 2 = 4×) and negative log10 p-value (e.g., 2 = p-value of 0.01).
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pone-0004486-g002: Gene expression for Single Blood isolate.Heat map for single blood isolate that was divided into four. RNA was purified, labeled and hybridized separately for each sample. Red is greater and green is less than the average gene specific fluorescence. First column (P) is the p-value from a one-way ANOVA. Only 6 genes (2.3%) out of 252 are significant at a critical p-value of 0.01. P-values (−log10) shown in the heat map are from an ANOVA for significant differences among samples using the 8 replicates for each separate RNA isolation. Color bar gives fold difference for log2 gene expression (e.g., 2 = 4×) and negative log10 p-value (e.g., 2 = p-value of 0.01).

Mentions: A one-way ANOVA was used to test for the technical variation in gene expression between the four RNA samples isolated from a single blood sample (Fig. 2). Among all 252 genes (eight replicates per gene per sample) only 6 genes were significantly different for the four isolates at a critical p-value of 0.01. Three false-positives are expected at a p-value of 0.01 and thus with only 6 significant differences (Fig. 2) there is little evidence that separate RNA isolation, labeling and hybridization has much affect on measures of gene expression. The lack of differences is not due to high technical variation: CV (standard deviation/mean) among the eight replicates was 4% and, only three genes had a CV of >10%. Nor was it due to the low p-value of 0.01 versus 0.05 (Fig. 2); the number of significant differences simply reflects the p-values.


Technical analysis of cDNA microarrays.

Scott CP, VanWye J, McDonald MD, Crawford DL - PLoS ONE (2009)

Gene expression for Single Blood isolate.Heat map for single blood isolate that was divided into four. RNA was purified, labeled and hybridized separately for each sample. Red is greater and green is less than the average gene specific fluorescence. First column (P) is the p-value from a one-way ANOVA. Only 6 genes (2.3%) out of 252 are significant at a critical p-value of 0.01. P-values (−log10) shown in the heat map are from an ANOVA for significant differences among samples using the 8 replicates for each separate RNA isolation. Color bar gives fold difference for log2 gene expression (e.g., 2 = 4×) and negative log10 p-value (e.g., 2 = p-value of 0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637424&req=5

pone-0004486-g002: Gene expression for Single Blood isolate.Heat map for single blood isolate that was divided into four. RNA was purified, labeled and hybridized separately for each sample. Red is greater and green is less than the average gene specific fluorescence. First column (P) is the p-value from a one-way ANOVA. Only 6 genes (2.3%) out of 252 are significant at a critical p-value of 0.01. P-values (−log10) shown in the heat map are from an ANOVA for significant differences among samples using the 8 replicates for each separate RNA isolation. Color bar gives fold difference for log2 gene expression (e.g., 2 = 4×) and negative log10 p-value (e.g., 2 = p-value of 0.01).
Mentions: A one-way ANOVA was used to test for the technical variation in gene expression between the four RNA samples isolated from a single blood sample (Fig. 2). Among all 252 genes (eight replicates per gene per sample) only 6 genes were significantly different for the four isolates at a critical p-value of 0.01. Three false-positives are expected at a p-value of 0.01 and thus with only 6 significant differences (Fig. 2) there is little evidence that separate RNA isolation, labeling and hybridization has much affect on measures of gene expression. The lack of differences is not due to high technical variation: CV (standard deviation/mean) among the eight replicates was 4% and, only three genes had a CV of >10%. Nor was it due to the low p-value of 0.01 versus 0.05 (Fig. 2); the number of significant differences simply reflects the p-values.

Bottom Line: Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization.Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression.However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida, United States of America.

ABSTRACT

Background: There is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization.

Methodology/principal findings: In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course.

Conclusion: There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise.

Show MeSH
Related in: MedlinePlus