Skvortsov Dmitriy, Abdueva Diana, Curtis Christina, Schaub Betty, Tavaré Simon
Department of Human Genetics, University of California Los Angeles, CA, USA.
Nucleic Acids Res. 2007;35(12):4154-63. doi: 10.1093/nar/gkm348. Epub 2007 Jun 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 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.
Affymetrix进行的微阵列杂交实验性掺入研究表明，荧光强度信号对靶标浓度呈非线性响应。已经提出了几种理论模型来解释这些数据。结果表明，朗缪尔吸附等温线概括了信号对浓度响应的一般趋势。然而，该模型无法解释观察到的信号的一些关键特性。特别是，根据简单的朗缪尔等温线，所有探针应以相同的强度水平饱和。然而，在公开可用的Affymetrix掺入数据集中未观察到这种效应。相反，发现饱和强度变化很大，并且可以基于探针序列组成进行预测。在我们的实验研究中，我们尝试使用定制的流体脚本来说明观察到的探针强度中无法解释的变化。我们通过实验探索严格洗涤、靶标浓度和杂交时间对最终微阵列信号的影响。通过在严格洗涤之前和之后扫描芯片来评估洗涤效果。对特异性和非特异性靶标的选择性标记允许可视化和研究特异性和非特异性信号成分的洗涤效果。我们提出了一种新的探针-靶标杂交机制定性模型，该模型与短寡核苷酸微阵列观察到的杂交和洗涤特性一致。这项研究表明，洗涤循环中未完全结合的靶标的解吸导致了观察到的饱和水平差异。
