Specificity assessment from fractionation experiments (SAFE): a novel method to evaluate microarray probe specificity based on hybridisation stringencies.
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.
Drobyshev
Alexei L
AL
Institute of Experimental Genetics, GSF-National Research Centre for Environment and Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
Machka
Christine
C
Horsch
Marion
M
Seltmann
Matthias
M
Liebscher
Volkmar
V
Hrabé de Angelis
Martin
M
Beckers
Johannes
J
D000818:Animals; D015342:DNA Probes / Q000235:genetics / Q000592:standards*; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013696:Temperature; D013737:Testis / Q000378:metabolism
D015342:DNA Probes; D005456:Fluorescent Dyes; D012313:RNA