Technical analysis of cDNA microarrays

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.

Figure 1. 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).

Figure 2. 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).

Figure 3. Individuals sampled over time.

Heat map for one individual (00) sampled 4 times over a total of 6 weeks. Numbers above the heat map are time points (0, 2, 4 & 6 weeks) and the “P” is for p-value (−log₁₀). P-values are from the ANOVA that tested for differences among separate blood isolations within an individual (4 isolations and 8 replicates per isolation). For gene expression, red is greater and green is lower expression than the mean expression for each gene. Table provides number of significant genes and percent (rounded up) out of the total of 304. 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).