Abstract
BackgroundPost-hybridization washing is an essential part of microarray experiments. Both the quality of the experimental washing protocol and adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities.ResultsWe conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of the number of washing cycles. For calibration and analysis of the intensity data we make use of the 'hook' method which allows intensity contributions due to non-specific and specific hybridization of perfect match (PM) and mismatch (MM) probes to be disentangled in a sequence specific manner. On average, washing according to the standard protocol removes about 90% of the non-specific background and about 30-50% and less than 10% of the specific targets from the MM and PM, respectively. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. It depends on the intensity contribution due to specific and non-specific hybridization per probe which can be estimated for each probe using existing methods. The washing function allows probe intensities to be calibrated for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures, especially in the limit of small and large values.ConclusionsWashing is among the factors which potentially distort expression measures. The proposed first-order correction method allows direct implementation in existing calibration algorithms for microarray data. We provide an experimental 'washing data set' which might be used by the community for developing amendments of the washing correction.
Highlights
Post-hybridization washing is an essential part of microarray experiments
Burden et al [9] showed that post hybridization washing explains the observed discrepancy: Sequencedependent dissociation of the probes in the absence of free targets decreases the amount of probe-bound targets differently for perfect match (PM) and MM probes, resulting in observed intensity differences
The standard protocol includes a low stringent wash (900 mM Na+) at 30°C followed by 6 stringent wash cycles at 50°C with decreased salt concentration (100 mM Na+). After these washes the array is stained with streptavidin phycoerythrin (SAPE) in two rounds which are intermitted by a round of anti-SAPE antibody staining and non-stringent washes
Summary
Post-hybridization washing is an essential part of microarray experiments. Both the quality of the experimental washing protocol and adequate consideration of washing in intensity calibration affect the quality of the expression estimates extracted from the microarray intensities. The washing step aims at improving the signal-to-noise ratio by removing free optical markers and hybridized Both the quality of washing achieved in the experimental protocol and the adequate consideration of the washing mechanism in intensity calibration affect the quality of the expression estimates extracted from the microarray intensities and subsequent downstream analysis. Burden et al [9] showed that post hybridization washing explains the observed discrepancy: Sequencedependent dissociation of the probes in the absence of free targets decreases the amount of probe-bound targets differently for PM and MM probes, resulting in observed intensity differences This hypothesis was later confirmed by the ‘washing’ experiments of Skvortsov et al [4] on Affymetrix GeneChip arrays. The authors applied customized fluidic scripts and selective labeling of specific and non-specific targets and measured the respective signal components prior to and after stringent washes to estimate the washing yield in dependence of the hybridization mode, target concentration and equilibration time prior to washing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
