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Explaining differences in saturation levels for Affymetrix GeneChip arrays.

ABSTRACT: The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in data sets. On the contrary, it was found that the saturation intensities vary greatly and can be predicted based on the probe sequence composition. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration and hybridization time on the final microarray signal. The washing effect is assessed by scanning chips both prior to and after the stringent wash. Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. We propose a new qualitative model of the probe-target hybridization mechanism that is in agreement with observed hybridization and washing properties of short oligonucleotide microarrays. This study demonstrates that desorption of incompletely bound targets during the washing cycle contributes to the observed difference in saturation levels.

SUBMITTER: Skvortsov D

PROVIDER: S-EPMC1919478 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Explaining differences in saturation levels for Affymetrix GeneChip arrays.

Skvortsov Dmitriy D Abdueva Diana D Curtis Christina C Schaub Betty B Tavaré Simon S

Nucleic acids research 20070612 12

The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isot ...[more]

PMID: 17567617

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Project description:BackgroundAffymetrix gene expression arrays incorporate paired perfect match (PM) and mismatch (MM) probes to distinguish true signals from those arising from cross-hybridization events. A MM signal often shows greater intensity than a PM signal; we propose that one underlying cause is the presence of allelic variants arising from single nucleotide polymorphisms (SNPs). To annotate and characterize SNP contributions to anomalous probe binding behavior we have developed a software tool called AffyMAPSDetector.ResultsAffyMAPSDetector can be used to describe any Affymetrix expression GeneChip with respect to SNPs. When AffyMAPSDetector was run on GeneChip HG-U95Av2 against dbSNP-build-123, we found 7286 probes (belonging to 2,582 probesets) containing SNPs, out of which 325 probes contained at least one SNP at position 13. Against dbSNP-build-126, 8758 probes (belonging to 3,002 probesets) contained SNPs, of which 409 probes contained at least one SNP at position 13. Therefore, depending on the expressed allele, the MM probe can sometimes be the transcript complement. This information was used to characterize probe measurements reported in a published, well-replicated lung adenocarcinoma study. The total intensity distributions showed that the SNP-containing probes had a larger negative mean intensity difference (PM-MM) and greater range of the difference than did probes without SNPs. In the sample replicates, SNP-containing probes with reproducible intensity ratios were identified, allowing selection of SNP probesets that yielded unique sample signatures. At the gene expression level, use of the (MM-PM) value for SNP-containing probes resulted in different Presence/Absence calls for some genes. Such a change in status of the genes has the clear potential for influencing downstream clustering and classification results.ConclusionOutput from this tool characterizes SNP-containing probes on GeneChip microarrays, thus improving our understanding of factors contributing to expression measurements. The pattern of SNP binding examined so far indicates distinct behavior of the SNP-containing probes and has the potential to help us identify new SNPs. Knowing which probes contain SNPs provides flexibility in determining whether to include or exclude them from gene-expression intensity calculations; selected sets of SNP-containing probes produce sample-unique signatures. AffyMAPSDetector information is available at http://www.binf.gmu.edu/weller/BMC_bioinformatics/AffyMapsDetector/index.html.

Dataset Information

Explaining differences in saturation levels for Affymetrix GeneChip arrays.

Publications

Explaining differences in saturation levels for Affymetrix GeneChip arrays.

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