A common reference for cDNA microarray hybridizations.
ABSTRACT Comparisons of expression levels across different cDNA microarray experiments are easier when a common reference is co-hybridized to every microarray. Often this reference consists of one experimental control sample, a pool of cell lines or a mix of all samples to be analyzed. We have developed an alternative common reference consisting of a mix of the products that are spotted on the array. Pooling part of the cDNA PCR products before they are printed and their subsequent amplification towards either sense or antisense cRNA provides an excellent common reference. Our results show that this reference yields a reproducible hybridization signal in 99.5% of the cDNA probes spotted on the array. Accordingly, a ratio can be calculated for every spot, and expression levels across different hybridizations can be compared. In dye-swap experiments this reference shows no significant ratio differences, with 95% of the spots within an interval of +/-0.2-fold change. The described method can be used in hybridizations with both amplified and non-amplified targets, is time saving and provides a constant batch of common reference that lasts for thousands of hybridizations.
Nucleic Acids Research 01/2003; 31(5):20e-20. DOI:10.1093/nar/gng020 · 8.81 Impact Factor
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ABSTRACT: An efficient spot-adaptive segmentation technique was developed by suitable combining in a cascade mode the benefits of image enhancement (Contrast Limited Adaptive Histogram Equalization technique (CLAHE)) and image segmentation (Seeded Region Growing technique (SRG)) in order to improve genes' quantification in microarray images. Microarrays utilized for evaluation purposes comprised 7 publicly available images. Initially, an image griding algorithm was employed to divide the image into rectangular image-cells. Subsequently, CLAHE was applied on each individual image-cell, initial SRG-seed was set at the image-cell's center, and SRG-threshold was estimated from the image-cell's background. The spot's boundary was referred to the corresponding cell spot in the original image and the spot's intensity was evaluated. Extracted intensities were comparatively evaluated against a well-established commercial software package (MAGIC TOOL) employing the Jeffrey's divergence-metric. The metric of the spot-adaptive segmentation technique was about double as compared to MAGIC TOOL's metric, with differences ranging between 1.23 and 5.21 in the processed images. Regarding processing time, the proposed method required half the time of MAGIC TOOL's (211 secs against 487 secs) to process the same cDNA image on the same computer.
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ABSTRACT: two. Our aim is to find D-optimal, A-optimal and related L-optimal designs for such experi-ments. Previous work has focussed on exhaustive search algorithms, which in many practical, large-scale experiments is infeasible. We propose using simulated annealing to search for near-optimal designs. We also consider a sub-class of designs, the so-called interwoven loop designs, which seem to possess good optimality properties. The conclusions are also relevant for certain quantitative comparative experiments. Keywords: microarray experiments, experimental design, simulated annealing, A-optimality, D-optimality, L-optimality