Sensitivity of 70-mer oligonucleotides and cDNAs for microarray analysis of gene expression in Arabidopsis and its related species

Abstract

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60-70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance (anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from approximately 26,000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids.

Figure 1

Bar plots showing the mean squares from analysis of variance for the RNA1 vs. RNA2, RNA1 vs. RNA3, RNA2 vs. RNA3, and RNA123 vs. RNA123 microarray experiments. The parameters in the models (and the degrees of freedom) were: S = slide (5), D = dye labelling (1), T = target (1), F = feature type (1), G = gene (183), and the interactions of these effects.

Figure 2

Bar plots showing the means squares from analysis of variance for the Arabidopsis arenosa and Brassica oleracea microarray experiments. The parameters in the models (and the degrees of freedom) were: S = slide (3), D = dye labelling (1), T = target (1), F = feature type (1), G = gene (183), and the interactions of these effects.

Figure 3

Bar plots showing the mean squares from analysis of variance of microarray experiments using 183 gene sequences and target RNAs isolated from Arabidopsis thaliana leaves and flower buds. (a) denatured cDNA and oligo features in the analysis, (b) denatured cDNA and oligo features analysed separately, and (c) scatter plot of mean log fold-changes (leaves /flower buds) for cDNA and oligo features analysed separately. (d), (e) and (f) are the same as above, but for sets of experiments with undenatured cDNA and oligo features. The parameters in the models (and the degrees of freedom) were: S = slide (5), D = dye labelling (1), T = target (1), F = feature type (1), G = gene (183), and the interactions of these effects.

Figure 3

Bar plots showing the mean squares from analysis of variance of microarray experiments using 183 gene sequences and target RNAs isolated from Arabidopsis thaliana leaves and flower buds. (a) denatured cDNA and oligo features in the analysis, (b) denatured cDNA and oligo features analysed separately, and (c) scatter plot of mean log fold-changes (leaves /flower buds) for cDNA and oligo features analysed separately. (d), (e) and (f) are the same as above, but for sets of experiments with undenatured cDNA and oligo features. The parameters in the models (and the degrees of freedom) were: S = slide (5), D = dye labelling (1), T = target (1), F = feature type (1), G = gene (183), and the interactions of these effects.

Figure 4

Bar plots comparing gene expression changes between Arabidopsis thaliana leaves and flower buds detected by microarrays with ‘undenatured’ cDNA features, undenatured oligo features, and quantitative RT-PCR (QRT-PCR). Results show fold-change of expression ratios (flower buds vs. leaves; F/L) obtained from six replications in the cDNA and oligo microarray and 3 replications from QRT-PCR, and are grouped into four categories: (a) similar results for cDNA, oligo and QRT-PCR (b) similar for oligos and QRT-PCR, but different for cDNA (c) different for cDNA and oligos, but similar for cDNA and QRT-PCR (d) similar result for cDNA and oligo but different for QRT-PCR.