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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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Linear relationship of RNA concentration to relative fluorescence.Graphs show linear relationship between concentrations of RNA (0.1–50×, A–C, and 0.1–10×, D–F) and relative fluorescence. Relative fluorescence is a normalized measure of fluorescence divided by the gene specific mean. 1× RNA is equal to 0.9 pmol/µl. Shown are the RNA concentrations versus fluorescence for 0.1 to 50× (A–C) and for 0.1× to 10× (D–F); for all genes (A and D), for the 78 genes with the highest R2 values (B and E), and for the 18 with lowest R2 values (C and F).
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pone-0004486-g001: Linear relationship of RNA concentration to relative fluorescence.Graphs show linear relationship between concentrations of RNA (0.1–50×, A–C, and 0.1–10×, D–F) and relative fluorescence. Relative fluorescence is a normalized measure of fluorescence divided by the gene specific mean. 1× RNA is equal to 0.9 pmol/µl. Shown are the RNA concentrations versus fluorescence for 0.1 to 50× (A–C) and for 0.1× to 10× (D–F); for all genes (A and D), for the 78 genes with the highest R2 values (B and E), and for the 18 with lowest R2 values (C and F).

Mentions: The linear relationship between the amount of RNA and relative fluorescence is shown in Figure 1. To remove the gene specific differences in expression, the fluorescence at each concentration was divided by the mean fluorescence for that specific gene (Fig. 1). The linear relationship between the amount of total fluorescent RNA added and the measures of gene specific fluorescence was determined for each gene. Most genes (176/212 or 83%) had an R2>95% and 78 genes had a nearly perfect R2 (>0.995; Fig. 1B; Table 3). Examining the 18 genes with the lowest R2 values (less than 0.8) revealed a non-linear relationship that can be explained by an apparent saturation at the 50× concentration of RNA (Fig. 1C). The relationship disappears if the fluorescence values for the 50× concentrations of RNA are removed and the 0.1 to 10× are plotted (Fig 1D–F). In the 100-fold range (0.1 to 10×) only three genes (1.4%) had R2 values less than 0.8 (Table 3). Examination of the higher concentrations (1.0 to 50×) revealed 19 genes (9%) with R2 less than 0.8 (Table 3). These data suggest that for most genes there is a linear relationship for a 500-fold range of RNA, however some cDNAs on the microarray will reach biological saturation at the highest RNA concentration.


Technical analysis of cDNA microarrays.

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

Linear relationship of RNA concentration to relative fluorescence.Graphs show linear relationship between concentrations of RNA (0.1–50×, A–C, and 0.1–10×, D–F) and relative fluorescence. Relative fluorescence is a normalized measure of fluorescence divided by the gene specific mean. 1× RNA is equal to 0.9 pmol/µl. Shown are the RNA concentrations versus fluorescence for 0.1 to 50× (A–C) and for 0.1× to 10× (D–F); for all genes (A and D), for the 78 genes with the highest R2 values (B and E), and for the 18 with lowest R2 values (C and F).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004486-g001: Linear relationship of RNA concentration to relative fluorescence.Graphs show linear relationship between concentrations of RNA (0.1–50×, A–C, and 0.1–10×, D–F) and relative fluorescence. Relative fluorescence is a normalized measure of fluorescence divided by the gene specific mean. 1× RNA is equal to 0.9 pmol/µl. Shown are the RNA concentrations versus fluorescence for 0.1 to 50× (A–C) and for 0.1× to 10× (D–F); for all genes (A and D), for the 78 genes with the highest R2 values (B and E), and for the 18 with lowest R2 values (C and F).
Mentions: The linear relationship between the amount of RNA and relative fluorescence is shown in Figure 1. To remove the gene specific differences in expression, the fluorescence at each concentration was divided by the mean fluorescence for that specific gene (Fig. 1). The linear relationship between the amount of total fluorescent RNA added and the measures of gene specific fluorescence was determined for each gene. Most genes (176/212 or 83%) had an R2>95% and 78 genes had a nearly perfect R2 (>0.995; Fig. 1B; Table 3). Examining the 18 genes with the lowest R2 values (less than 0.8) revealed a non-linear relationship that can be explained by an apparent saturation at the 50× concentration of RNA (Fig. 1C). The relationship disappears if the fluorescence values for the 50× concentrations of RNA are removed and the 0.1 to 10× are plotted (Fig 1D–F). In the 100-fold range (0.1 to 10×) only three genes (1.4%) had R2 values less than 0.8 (Table 3). Examination of the higher concentrations (1.0 to 50×) revealed 19 genes (9%) with R2 less than 0.8 (Table 3). These data suggest that for most genes there is a linear relationship for a 500-fold range of RNA, however some cDNAs on the microarray will reach biological saturation at the highest RNA concentration.

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