Microarray Image Analysis: Background Estimation using Region and Filtering Techniques

This report examines properties of two main methods used for local background estimation in microarray images. The first method uses samples from local regions defined such that they supposedly contain only background pixels; we call this a region method. This method is implemented in the Axon GenePix Pro software. The second method uses filtering techniques that removes the bright spots from the image. A version using morphological filters is implemented in the CSIRO Spot software. Using cDNA microarray slides, a detailed study and comparison of the background estimates of the Axon GenePix and the CSIRO Spot software have been performed. We have found that both software give a bias both within and between the two channels as well as having great variability. For the region method the number of background pixels determines the variance of the estimate while the distribution determines the bias. For the morphological method the number of background pixels used as well as the distribution of these pixels determines the level of the estimate and the variance. The morphological filtering method has been extended to the generalization rank filtering. For comparison, a region method with fixed number of pixels in the local background sample has been implemented. Both the fixed region method and the rank filtering implementations have been proven to greatly improve performance over the current Axon GenePix and CSIRO Spot implementations, reducing bias as well as variability significantly. This is especially important for low intensity spots where even a small bias has great influence.