Ratio-intensity plots before and after normalization. (A) log-transformed signal ratios (Cy3/Cy5) are plotted against the log-transformed products of signal intensities. Grey spots: discarded data that were filtered out by initial data processing (see text for details); blue spots: data used for further evaluation. Left side: temperature experiment with sample from cells grown at 25°C labelled with Cy5 and sample from cells grown at 30°C labelled with Cy3. Right side: identical sample labelled with Cy3 and Cy5 and hybridised on same array ('self-self' hybridisation): all signal ratios are expected to be 1, and the absence of differential gene expression is reflected by a tighter distribution of the spots. The number of the blue spots that were retained for data evaluation is 9161 (left) and 8560 (right). (B) As in (A) after normalization of the data using our local normalization scheme. (C) Overlay of spots before (blue) and after (red) normalization.