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.

Interpreting physiological responses to environmental change through gene expression profiling

“…These elements may lead to cross-hybridization between the cDNA and transcripts other than the target transcript. The degree to which these non-specific hybridization signals compromise array analysis is still unclear, but it appears that increasing the stringency of both hybridization and wash conditions can alleviate these problems (Drobyshev et al, 2003;Korkola et al, 2003). On the other hand, the tolerance of long cDNAs to base-pair mismatches can be turned into an advantage, since it allows nucleic acids from related taxa to be hybridized heterologously to a single species array (Renn et al, 2004;von Schalburg et al, 2005).…”

Reconstruction of noisy signals by minimization of non-convex functionals

“…A second domain is defined by PWS signals corrupted with White Gaussian noise, both alone and jointly with Impulsive noise. These denoising problems might result from scientific and engineering areas such as cross-hybridization of DNA [24], molecular bioscience [25], reconstruction of brain stimuli [26], among others. From a practical point of view, impulsive noises may appear due to sensor malfunctioning, while White Gaussian noise is mainly caused by poor illumination, high temperature, transmission errors, among others.…”

Functional genomic resources for potato

“…Recommendations with this platform include using linearly amplified cRNA as a target instead of singlestranded cDNA, and a specialized hybridization chamber to increase hybridization sensitivity and reproducibility. Agilent has identified ozone as a source of degradation of the Cy5 dye (Fare et al 2003), so, to address this, they offer specialized stabilization and drying solution to prevent this ozone-mediated degradation.…”

“…To evaluate probe specificity. Drobyshev et al (2003) suggested that the microarray could be subjected to a series of stringent washes that could differentiate between perfect and less than perfect hybridizations, but there has been no report of applying this approach to potato gene microarrays.…”