Spotting effect in microarray experiments

Abstract

Background: Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array.

Results: Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure.

Conclusions: The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis.

Figure 1

Spatial distribution of the signal for the self-hybridized Arabidopsis slide Each pixel represents the uncorrected log-ratio of the median Cy5 (635 nm) and Cy3 (532 nm) channel fluorescence measurements, associated to a printed DNA feature. Background is not represented. The picture is not a re-plot of the original image captured during the scanning process. Labels correspond to the 9-quantiles of the signal distribution.

Figure 2

Variogram of the signal by row for the Arabidopsis slide, before normalization

Figure 3

Variogram of the intensity (log(R × G)) by row for the Arabidopsis slide

Figure 4

Variogram for the raw signal by row (A) Tor270 slide; (B) Zhu473 slide; (C) Lieb3727 slide. No normalization is performed.

Figure 5

Distribution of the residuals (i.e. corrected signal) after reference normalization, for the Arabidopsis slide Labels correspond to the 9-quantiles of the residuals distribution.

Figure 6

Variogram of the residuals (i.e. corrected signal) after reference normalization, for the Arabidopsis slide