Dataset Information

Specificity assessment from fractionation experiments (SAFE): a novel method to evaluate microarray probe specificity based on hybridisation stringencies.

ABSTRACT: The cDNA-chip technology is a highly versatile tool for the comprehensive analysis of gene expression at the transcript level. Although it has been applied successfully in expression profiling projects, there is an ongoing dispute concerning the quality of such expression data. The latter critically depends on the specificity of hybridisation. SAFE (specificity assessment from fractionation experiments) is a novel method to discriminate between non- specific cross-hybridisation and specific signals. We applied in situ fractionation of hybridised target on DNA-chips by means of repeated washes with increasing stringencies. Different fractions of hybridised target are washed off at defined stringencies and the collected fluorescence intensity data at each step comprise the fractionation curve. Based on characteristic features of the fractionation curve, unreliable data can be filtered and eliminated from subsequent analyses. The approach described here provides a novel experimental tool to identify probes that produce specific hybridisation signals in DNA-chip expression profiling approaches. The iterative use of the SAFE procedure will result in increasingly reliable sets of probes for microarray experiments and significantly improve the overall efficiency and reliability of RNA expression profiling data from DNA-chip experiments.

SUBMITTER: Drobyshev AL

PROVIDER: S-EPMC140526 | biostudies-literature | 2003 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Specificity assessment from fractionation experiments (SAFE): a novel method to evaluate microarray probe specificity based on hybridisation stringencies.

Drobyshev Alexei L AL Machka Christine C Horsch Marion M Seltmann Matthias M Liebscher Volkmar V Hrabé de Angelis Martin M Beckers Johannes J

Nucleic acids research 20030101 2

The cDNA-chip technology is a highly versatile tool for the comprehensive analysis of gene expression at the transcript level. Although it has been applied successfully in expression profiling projects, there is an ongoing dispute concerning the quality of such expression data. The latter critically depends on the specificity of hybridisation. SAFE (specificity assessment from fractionation experiments) is a novel method to discriminate between non- specific cross-hybridisation and specific sign ...[more]

PMID: 12527790

Similar Datasets

Project description:BackgroundAlthough microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance.ResultsTwo custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements.ConclusionThe assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations.

Dataset Information

Specificity assessment from fractionation experiments (SAFE): a novel method to evaluate microarray probe specificity based on hybridisation stringencies.

Publications

Specificity assessment from fractionation experiments (SAFE): a novel method to evaluate microarray probe specificity based on hybridisation stringencies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets