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

Binder H, Krohn K, Preibisch S - Algorithms Mol Biol (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany. binder@izbi.uni-leipzig.de

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.

No MeSH data available.


Related in: MedlinePlus

Hook-characteristics of GeneChips of different generations (see figure, from left to the right). The chips are hybridized with mRNA extracts from tumour samples (thyroid nodules, two parts on the left; [9] and references cited therein) and from the Universal Human Reference RNA (chips c and d; see [10] for details). The figures show the raw hook (below), the corrected hook (middle), the probability density distribution (middle, right axis) and the theoretical curve fitted of the mix-, S- and sat-ranges of the corrected hook curves (above). The percentage of absent probes (%N) is given within the figures.
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Figure 3: Hook-characteristics of GeneChips of different generations (see figure, from left to the right). The chips are hybridized with mRNA extracts from tumour samples (thyroid nodules, two parts on the left; [9] and references cited therein) and from the Universal Human Reference RNA (chips c and d; see [10] for details). The figures show the raw hook (below), the corrected hook (middle), the probability density distribution (middle, right axis) and the theoretical curve fitted of the mix-, S- and sat-ranges of the corrected hook curves (above). The percentage of absent probes (%N) is given within the figures.

Mentions: Figure 3 shows a collection of representative hook-curves taken from four hybridizations of human-genome chips of different generations. Along the chip generations the spot-size of the probes decreases from 20 μm (U95), over 18 μm (U133A) to 11 μm (U133-plus2). The reduction of spot-size has enabled to increase the number of probe sets per chip from 16.000 over 22.000 to 54.000, respectively [6,7]. In addition, this development is accompanied by modifications of the reagent-kits and the scanning technique [7,8]. Importantly, also probe design and selection have been improved by applying more sophisticated genomic and thermodynamic criteria especially for chip generations following the U95. Chip data shown in Figure 3 refer to RNA prepared from tissue samples (thyroid nodules; [9]) and to Universal Human Reference RNA [10].


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

Binder H, Krohn K, Preibisch S - Algorithms Mol Biol (2008)

Hook-characteristics of GeneChips of different generations (see figure, from left to the right). The chips are hybridized with mRNA extracts from tumour samples (thyroid nodules, two parts on the left; [9] and references cited therein) and from the Universal Human Reference RNA (chips c and d; see [10] for details). The figures show the raw hook (below), the corrected hook (middle), the probability density distribution (middle, right axis) and the theoretical curve fitted of the mix-, S- and sat-ranges of the corrected hook curves (above). The percentage of absent probes (%N) is given within the figures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543012&req=5

Figure 3: Hook-characteristics of GeneChips of different generations (see figure, from left to the right). The chips are hybridized with mRNA extracts from tumour samples (thyroid nodules, two parts on the left; [9] and references cited therein) and from the Universal Human Reference RNA (chips c and d; see [10] for details). The figures show the raw hook (below), the corrected hook (middle), the probability density distribution (middle, right axis) and the theoretical curve fitted of the mix-, S- and sat-ranges of the corrected hook curves (above). The percentage of absent probes (%N) is given within the figures.
Mentions: Figure 3 shows a collection of representative hook-curves taken from four hybridizations of human-genome chips of different generations. Along the chip generations the spot-size of the probes decreases from 20 μm (U95), over 18 μm (U133A) to 11 μm (U133-plus2). The reduction of spot-size has enabled to increase the number of probe sets per chip from 16.000 over 22.000 to 54.000, respectively [6,7]. In addition, this development is accompanied by modifications of the reagent-kits and the scanning technique [7,8]. Importantly, also probe design and selection have been improved by applying more sophisticated genomic and thermodynamic criteria especially for chip generations following the U95. Chip data shown in Figure 3 refer to RNA prepared from tissue samples (thyroid nodules; [9]) and to Universal Human Reference RNA [10].

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany. binder@izbi.uni-leipzig.de

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.

No MeSH data available.


Related in: MedlinePlus