"Hook"-calibration of GeneChip-microarrays: Chip characteristics and expression measures

Hans Binder¹ , Knut Krohn² and Stephan Preibisch³

¹Interdisciplinary Centre for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany

²Interdisciplinary Center for Clinical Research, Medical Faculty; University of Leipzig, D-04107 Leipzig, Germany

³Max-Planck-Institute for Molecular Cell Biology and Genetics, D-01307 Dresden, Germany

author email corresponding author email

Algorithms for Molecular Biology 2008, 3:11doi:10.1186/1748-7188-3-11


Published:	29 August 2008

Abstract

Background

Microarray experiments rely on several critical steps that may introduce biases and uncertainty in downstream analyses. These steps include mRNA sample extraction, amplification and labelling, hybridization, and scanning causing chip-specific systematic variations on the raw intensity level. Also the chosen array-type and the up-to-dateness of the genomic information probed on the chip affect the quality of the expression measures. In the accompanying publication we presented theory and algorithm of the so-called hook method which aims at correcting expression data for systematic biases using a series of new chip characteristics.

Results

In this publication we summarize the essential chip characteristics provided by this method, analyze special benchmark experiments to estimate transcript related expression measures and illustrate the potency of the method to detect and to quantify the quality of a particular hybridization. It is shown that our single-chip approach provides expression measures responding linearly on changes of the transcript concentration over three orders of magnitude. In addition, the method calculates a detection call judging the relation between the signal and the detection limit of the particular measurement. The performance of the method in the context of different chip generations and probe set assignments is illustrated. The hook method characterizes the RNA-quality in terms of the 3'/5'-amplification bias and the sample-specific calling rate. We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes. The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.

Conclusion

The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.