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.

Figure 1.

Adsorption isotherms observed for Affymetrix microarrays. The observed adsorption isotherms (signal intensity versus target concentration) for Affymetrix microarrays exhibit different saturation intensities on both the log2 (left panel) and natural (right panel) scale.

Figure 2.

Standard Affymetrix eukaryotic protocol for hybridization, washing, staining and scanning (left) and our modified protocol (right).

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.

Figure 4.

Scatterplot of intensities for corresponding probes resulting from the standard and modified protocols. Shown are the log₂ intensities for spiked clones after the stringent wash from a chip processed according to the standard Affymetrix protocol versus the log₂ intensities of the same probes on a chip that was processed according to the altered protocol; PM signal [red] and MM signal [blue].

Figure 5.

Washing effect. (A) The log₂ observed intensities for spiked targets in the presence of complex background before the wash for PM (solid black) and MM (dashed black) probes, and after the wash for PM (solid gray) and MM (dashed gray) probes. Intensities are ordered by the pre-wash intensity of PM and MM probes. (B) The observed wash effect (pre/post wash intensity ratio) for spiked probes in the presence of complex background versus the log₂ observed PM pre-wash intensity. Black and gray dots represent the PM wash effect after the first and second wash, respectively. Open black and gray circles represent the MM wash effect after the first and second wash, respectively.

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.

Figure 7.

Concentration effect. (A) The comparison of intensity ratios between hybridization experiments with clone concentrations of 1 nM and 10 nM for PM (black circles) and MM (open circles) probes versus the log₂ intensity before the wash. (B) The observed log₂ difference in the wash effect for 1 and 10 nM for PM (black circles) and MM (open circles) probes versus the log 2 intensity before the wash.

Figure 8.

Hybridization time effect. (A) The comparison of intensity ratios for spiked probes following 16 and 40 h of hybridization for PM (solid circles) and MM (open circles) probes. (B) shows the log₂ difference in the wash effect for hybridization experiments with 16 and 40 h hybridization times for PM (solid circles) and MM (open circles) probes.