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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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Numerical solution of system (3). X-axis represents time in hours, Y-axis represents the fraction of chip that is occupied by particular targets. The colored lines represent the following: black, the fraction of unoccupied oligos; green, the fraction of oligos occupied by non-specifically absorbed targets; blue, the fraction occupied by intermediate products (partially zipped complexes with varying degrees of completion); orange, the fraction of all oligos bound to specific targets (fully and partially zipped); red, the fraction occupied by complete fully zipped duplexes.
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Figure 3: Numerical solution of system (3). X-axis represents time in hours, Y-axis represents the fraction of chip that is occupied by particular targets. The colored lines represent the following: black, the fraction of unoccupied oligos; green, the fraction of oligos occupied by non-specifically absorbed targets; blue, the fraction occupied by intermediate products (partially zipped complexes with varying degrees of completion); orange, the fraction of all oligos bound to specific targets (fully and partially zipped); red, the fraction occupied by complete fully zipped duplexes.

Mentions: The described model predicts that for short hybridization times there is a population of incompletely bound duplexes as well as a number of fully zipped duplexes and that over time the number of fully zipped pairs increases. Figure 3 illustrates this model and shows probe occupation with different types of duplexes as a function of time. Initially, non-specific targets (green line) quickly occupy a significant fraction of probes. This is followed by accumulation of specifically bound probe : target duplexes (blue line). The accumulation of fully zipped duplexes (red line) is delayed by the time approximately equal to the sum of relaxation times of all of the intermediate steps. The sum of all intermediate products except for the non-specifically adsorbed targets is shown in orange. Thus, subjecting the chip to a stringent wash cycle is roughly equivalent to shifting from the orange to the red curve; see Figure 3.Figure 3.


Explaining differences in saturation levels for Affymetrix GeneChip arrays.

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

Numerical solution of system (3). X-axis represents time in hours, Y-axis represents the fraction of chip that is occupied by particular targets. The colored lines represent the following: black, the fraction of unoccupied oligos; green, the fraction of oligos occupied by non-specifically absorbed targets; blue, the fraction occupied by intermediate products (partially zipped complexes with varying degrees of completion); orange, the fraction of all oligos bound to specific targets (fully and partially zipped); red, the fraction occupied by complete fully zipped duplexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Numerical solution of system (3). X-axis represents time in hours, Y-axis represents the fraction of chip that is occupied by particular targets. The colored lines represent the following: black, the fraction of unoccupied oligos; green, the fraction of oligos occupied by non-specifically absorbed targets; blue, the fraction occupied by intermediate products (partially zipped complexes with varying degrees of completion); orange, the fraction of all oligos bound to specific targets (fully and partially zipped); red, the fraction occupied by complete fully zipped duplexes.
Mentions: The described model predicts that for short hybridization times there is a population of incompletely bound duplexes as well as a number of fully zipped duplexes and that over time the number of fully zipped pairs increases. Figure 3 illustrates this model and shows probe occupation with different types of duplexes as a function of time. Initially, non-specific targets (green line) quickly occupy a significant fraction of probes. This is followed by accumulation of specifically bound probe : target duplexes (blue line). The accumulation of fully zipped duplexes (red line) is delayed by the time approximately equal to the sum of relaxation times of all of the intermediate steps. The sum of all intermediate products except for the non-specifically adsorbed targets is shown in orange. Thus, subjecting the chip to a stringent wash cycle is roughly equivalent to shifting from the orange to the red curve; see Figure 3.Figure 3.

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