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Comparative Study
. 2004 Feb 13;32(3):e29.
doi: 10.1093/nar/gnh027.

Reliability of gene expression ratios for cDNA microarrays in multiconditional experiments with a reference design

Rainer König  1 , Danila Baldessari, Nicolas Pollet, Christof Niehrs, Roland Eils
Affiliations
Comparative Study

Reliability of gene expression ratios for cDNA microarrays in multiconditional experiments with a reference design

Rainer König et al. Nucleic Acids Res. .

Abstract

In a typical gene expression profiling experiment with multiple conditions, a common reference sample is used for co-hybridization with the samples to yield expression ratios. Differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. In this study we test the validity of this approach. Differential expression of a sample pair (i, j) was obtained in two ways: directly, by hybridizations of sample i versus j, and indirectly, by multiplying the expression ratios for hybridizations of sample i versus pool and pool versus sample j. We performed gene expression profiling using amphibian embryos (Xenopus laevis). Every sample combination of four different stages and a pool was profiled. Direct and indirect values were compared and used as the quality criterion for the data. Based on this criterion, 82% of all ratios were found to be sufficiently accurate. To increase the reliability of the signals, several widely used filtering techniques were tested. Filtering by differences of repeated hybridizations was found to be the optimal filter. Finally, we compared microarray-based gene expression profiles with the corresponding expression patterns obtained by whole-mount in situ hybridizations, resulting in a 90% correspondence.

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Figures

Figure 1
Figure 1
Experimental set-up. All possible pairs of conditions (developmental stages 10.5, 13, 19, 38 and a pool) were hybridized, yielding 10 different direct ratios r10,19, r13,38 etc. Each hybridization was repeated in colour-reverse mode.
Figure 2
Figure 2
Indirect versus direct log2-ratios, plotted for each of the six possible sample pairs. Each spot represents a clone. Note that, under ideal conditions, indirect and direct ratios should be equal and all spots should be located on the 45° diagonal.
Figure 3
Figure 3
Indirect versus direct log2-ratios for all clones and sample pairs. Dark spots represent good quality values and light spots poor quality values according to our quality criterion (see Materials and Methods) Good quality values are grouped in three blocks corresponding to categories ‘up’ (upper, right), ‘not-changed’ (centred) and ‘down’ (lower, left).
Figure 4
Figure 4
Percentages of good quality values are shown at different filtering stringencies. Here, a filter stringency of 0 corresponds to no filtering (i.e. all expression values are retained), and 80 corresponds to 80% of all values are discarded due to the applied signal validation technique. The signal validation technique that uses differences of repeated hybridizations (hd) performed best, as noted by a considerably steeper increase for stringencies larger than 30%. The other filters are based on differences of double spots (dds), differences between mean and median of the pixels for a spot (dmm) and signal intensities (si), respectively.
Figure 5
Figure 5
Performance of the filters when taking only expression values that showed changes in expression (only ‘up’ and ‘down’ categories, neglecting ‘not-changed’ values) Axes and labelling as in Figure 4.
Figure 6
Figure 6
Comparison of differential expression obtained by our microarray study (columns 3 and 4) and in situ hybridizations from previous work (22), exemplified for six clones. Microarray-based differential expression is encoded by 1 for upregulated, –1 for downregulated and 0 for not-changed. For details see Results. Note that, for example, 10→13 specifies expressions of 13/10.
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