Explaining differences in saturation levels for Affymetrix GeneChip arrays.
Explaining differences in saturation levels for Affymetrix GeneChip arrays.
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DOI：
10.1093/nar/gkm348
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发表时间：
2007
影响因子：
14.9
通讯作者：
Tavaré S
中科院分区：
文献类型：
--
作者：
Skvortsov D;Abdueva D;Curtis C;Schaub B;Tavaré S
由Affytek进行的微阵列杂交的实验性加标研究证明了荧光强度信号对靶浓度的非线性响应。已经提出了几个理论模型来解释这些数据。结果表明，Langmuir吸附等温线概括了信号对浓度的响应的一般趋势。然而，该模型未能解释观测信号的一些关键特性。特别地，根据简单的朗缪尔等温线，所有探针应在相同的强度水平下饱和。然而，在公开可用的Affyoung加标数据集中未观察到该效应。相反，发现饱和强度变化很大，并且可以基于探针序列组成来预测。在我们的实验研究中，我们试图使用定制的射流脚本来解释所观察到的探针强度的无法解释的变化。我们通过实验探索了严格洗涤、靶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。特异性和非特异性靶标的选择性标记允许对特异性和非特异性信号组分的洗涤效果进行可视化和研究。我们提出了一个新的定性模型的探针-靶杂交机制，这是在短寡核苷酸微阵列观察到的杂交和洗涤性能的协议。该研究表明，在洗涤循环期间未完全结合的靶标的解吸有助于观察到的饱和度差异。
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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DOI：
10.1073/pnas.1534744100
发表时间：
2003-09-30
影响因子：
11.1
作者：
Mei, R;Hubbell, E;Webster, TA
通讯作者：
Webster, TA
影响因子：
14.9
作者：
Peterson, AW;Heaton, RJ;Georgiadis, RM
通讯作者：
Georgiadis, RM
影响因子：
14.9
作者：
Gao Y;Wolf LK;Georgiadis RM
通讯作者：
Georgiadis RM
影响因子：
2.7
作者：
Burden, Conrad J.;Pittelkow, Yvonne;Wilson, Susan R.
通讯作者：
Wilson, Susan R.
影响因子：
15
作者：
Peterson, AW;Wolf, LK;Georgiadis, RM
通讯作者：
Georgiadis, RM