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Explaining differences in saturation levels for Affymetrix GeneChip arrays.

Skvortsov D, Abdueva D, Curtis C, Schaub B, Tavaré S - Nucleic Acids Res. (2007)

Bottom Line: 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.The washing effect is assessed by scanning chips both prior to and after the stringent wash.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of California Los Angeles, CA, USA.

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.

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Washing effect for specific and non-specific signal. (A) A boxplot of non-specifically bound probes intensities before and after the stringent wash. (B) before- versus after-wash scatterplot of specifically and non-specifically bound probe intensities; gray dots represent non-specific and black circles represent specific probe intensities.
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Figure 6: Washing effect for specific and non-specific signal. (A) A boxplot of non-specifically bound probes intensities before and after the stringent wash. (B) before- versus after-wash scatterplot of specifically and non-specifically bound probe intensities; gray dots represent non-specific and black circles represent specific probe intensities.

Mentions: Selective biotin-labeling of spiked clones and non-specific complex background RNA enabled us to explore the properties of specifically and non-specifically formed duplexes. Figure 6 presents a comparison of the signal behavior of specifically and non-specifically bound targets. Figure 6A shows a boxplot of non-specifically bound probe intensities before and after the stringent wash. Prior to wash, the majority of non-specific probes demonstrate a noticeable signal of 7–9 on the log2 scale (or 130–500 on natural scale), while after the stringent wash, the interquantile range shifts to the 5–6 range on the log2 scale (or 30–60 on natural scale), corresponding to nearly ‘optical background’ level. Figure 6B offers a superimposed scatterplot of specifically and non-specifically bound probe intensities before and after the stringent wash. Examination of this plot reveals that a small fraction of non-specific probes generates a high response, comparable to the intensity of the specific probes, in both pre- and post-wash conditions. Similarly, a fraction of probes representing specifically bound targets demonstrate a low signal, comparable to non-specifically bound or non-responding probe intensities. We explain these observations by significant cross-hybridization in the first case and presence of non-responding probes and/or probes with no complimentary match in target sequences in the second case. Overlaying before-versus-after wash intensities for specific and non-specific target signals in Figure 6, we observe that the washing properties appear to be uniform across both specifically and non-specifically bound probes. This allows us to conclude that the hybridization/washing mechanism for both specific and non-specific targets is the same and the relationship between pre- and post-wash intensities is universal for all duplexes.Figure 6.


Explaining differences in saturation levels for Affymetrix GeneChip arrays.

Skvortsov D, Abdueva D, Curtis C, Schaub B, Tavaré S - Nucleic Acids Res. (2007)

Washing effect for specific and non-specific signal. (A) A boxplot of non-specifically bound probes intensities before and after the stringent wash. (B) before- versus after-wash scatterplot of specifically and non-specifically bound probe intensities; gray dots represent non-specific and black circles represent specific probe intensities.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919478&req=5

Figure 6: Washing effect for specific and non-specific signal. (A) A boxplot of non-specifically bound probes intensities before and after the stringent wash. (B) before- versus after-wash scatterplot of specifically and non-specifically bound probe intensities; gray dots represent non-specific and black circles represent specific probe intensities.
Mentions: Selective biotin-labeling of spiked clones and non-specific complex background RNA enabled us to explore the properties of specifically and non-specifically formed duplexes. Figure 6 presents a comparison of the signal behavior of specifically and non-specifically bound targets. Figure 6A shows a boxplot of non-specifically bound probe intensities before and after the stringent wash. Prior to wash, the majority of non-specific probes demonstrate a noticeable signal of 7–9 on the log2 scale (or 130–500 on natural scale), while after the stringent wash, the interquantile range shifts to the 5–6 range on the log2 scale (or 30–60 on natural scale), corresponding to nearly ‘optical background’ level. Figure 6B offers a superimposed scatterplot of specifically and non-specifically bound probe intensities before and after the stringent wash. Examination of this plot reveals that a small fraction of non-specific probes generates a high response, comparable to the intensity of the specific probes, in both pre- and post-wash conditions. Similarly, a fraction of probes representing specifically bound targets demonstrate a low signal, comparable to non-specifically bound or non-responding probe intensities. We explain these observations by significant cross-hybridization in the first case and presence of non-responding probes and/or probes with no complimentary match in target sequences in the second case. Overlaying before-versus-after wash intensities for specific and non-specific target signals in Figure 6, we observe that the washing properties appear to be uniform across both specifically and non-specifically bound probes. This allows us to conclude that the hybridization/washing mechanism for both specific and non-specific targets is the same and the relationship between pre- and post-wash intensities is universal for all duplexes.Figure 6.

Bottom Line: 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.The washing effect is assessed by scanning chips both prior to and after the stringent wash.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of California Los Angeles, CA, USA.

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.

Show MeSH