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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.


Affymetrix spiked-in experiment: The upper panel shows the hook obtained from one chip of this series. The predominant number of probes is hybridized with RNA of a HeLa-cell extract which was added to the chips to mimic a complex hybridization background (thick blue curve). The spike-probe sets are indicated by the open symbols and the respective transcript concentrations (see the numbers, the concentrations are given in units of pM). The horizontal distance between a spike position and the end point is related to the logarithm of the specific binding strength. The turning point between the N- and the mix-ranges defines the threshold for present probes. The dashed line is the fit of the Langmuir hybridization model to the data. The middle and lower parts show present/absent characteristics and the S/N-ratio of the spikes, respectively. The fraction of absent probes and the S/N ratio were calculated as mean values over all 42 chips of the experimental series (see thick lines). The open circles in the lower part show the individual probe-set values and thus the scatter of these points about their mean value. Spiked probes with nominal concentrations larger than 2 pM are "safely" called present. The S/N-ratio linearly correlates with the spiked-in concentration. The right axis of the lower part scales the expression estimates in units of the binding strength. The green dashed lines indicated that the threshold for calling probes as present corresponds to S/N-ratios R ≈ 0.1 – 2 and the S-binding strength of XN ≈ (0.5 – 5) 10-3.
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Figure 6: Affymetrix spiked-in experiment: The upper panel shows the hook obtained from one chip of this series. The predominant number of probes is hybridized with RNA of a HeLa-cell extract which was added to the chips to mimic a complex hybridization background (thick blue curve). The spike-probe sets are indicated by the open symbols and the respective transcript concentrations (see the numbers, the concentrations are given in units of pM). The horizontal distance between a spike position and the end point is related to the logarithm of the specific binding strength. The turning point between the N- and the mix-ranges defines the threshold for present probes. The dashed line is the fit of the Langmuir hybridization model to the data. The middle and lower parts show present/absent characteristics and the S/N-ratio of the spikes, respectively. The fraction of absent probes and the S/N ratio were calculated as mean values over all 42 chips of the experimental series (see thick lines). The open circles in the lower part show the individual probe-set values and thus the scatter of these points about their mean value. Spiked probes with nominal concentrations larger than 2 pM are "safely" called present. The S/N-ratio linearly correlates with the spiked-in concentration. The right axis of the lower part scales the expression estimates in units of the binding strength. The green dashed lines indicated that the threshold for calling probes as present corresponds to S/N-ratios R ≈ 0.1 – 2 and the S-binding strength of XN ≈ (0.5 – 5) 10-3.

Mentions: Figure 5 and Figure 6 show the hook curves, the absent calls and concentration measures of two special benchmark experiments. In the GeneLogic dilution series, cRNA from human liver tissue was hybridized on HG-U95 GeneChips in various amounts [1]. The decrease of the degree of non-specific binding upon dilution widens the horizontal dimension of the hook curve (see upper panel in Figure 5). Dilution decreases the concentration of specific and non-specific transcripts in a parallel fashion leaving their concentration ratio virtually constant. As expected, the S/N-ratio R of selected probes remains essentially constant whereas the binding strength of specific binding progressively decreases (compare solid symbols and thick lines in the lower panel of Figure 5).


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

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

Affymetrix spiked-in experiment: The upper panel shows the hook obtained from one chip of this series. The predominant number of probes is hybridized with RNA of a HeLa-cell extract which was added to the chips to mimic a complex hybridization background (thick blue curve). The spike-probe sets are indicated by the open symbols and the respective transcript concentrations (see the numbers, the concentrations are given in units of pM). The horizontal distance between a spike position and the end point is related to the logarithm of the specific binding strength. The turning point between the N- and the mix-ranges defines the threshold for present probes. The dashed line is the fit of the Langmuir hybridization model to the data. The middle and lower parts show present/absent characteristics and the S/N-ratio of the spikes, respectively. The fraction of absent probes and the S/N ratio were calculated as mean values over all 42 chips of the experimental series (see thick lines). The open circles in the lower part show the individual probe-set values and thus the scatter of these points about their mean value. Spiked probes with nominal concentrations larger than 2 pM are "safely" called present. The S/N-ratio linearly correlates with the spiked-in concentration. The right axis of the lower part scales the expression estimates in units of the binding strength. The green dashed lines indicated that the threshold for calling probes as present corresponds to S/N-ratios R ≈ 0.1 – 2 and the S-binding strength of XN ≈ (0.5 – 5) 10-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 6: Affymetrix spiked-in experiment: The upper panel shows the hook obtained from one chip of this series. The predominant number of probes is hybridized with RNA of a HeLa-cell extract which was added to the chips to mimic a complex hybridization background (thick blue curve). The spike-probe sets are indicated by the open symbols and the respective transcript concentrations (see the numbers, the concentrations are given in units of pM). The horizontal distance between a spike position and the end point is related to the logarithm of the specific binding strength. The turning point between the N- and the mix-ranges defines the threshold for present probes. The dashed line is the fit of the Langmuir hybridization model to the data. The middle and lower parts show present/absent characteristics and the S/N-ratio of the spikes, respectively. The fraction of absent probes and the S/N ratio were calculated as mean values over all 42 chips of the experimental series (see thick lines). The open circles in the lower part show the individual probe-set values and thus the scatter of these points about their mean value. Spiked probes with nominal concentrations larger than 2 pM are "safely" called present. The S/N-ratio linearly correlates with the spiked-in concentration. The right axis of the lower part scales the expression estimates in units of the binding strength. The green dashed lines indicated that the threshold for calling probes as present corresponds to S/N-ratios R ≈ 0.1 – 2 and the S-binding strength of XN ≈ (0.5 – 5) 10-3.
Mentions: Figure 5 and Figure 6 show the hook curves, the absent calls and concentration measures of two special benchmark experiments. In the GeneLogic dilution series, cRNA from human liver tissue was hybridized on HG-U95 GeneChips in various amounts [1]. The decrease of the degree of non-specific binding upon dilution widens the horizontal dimension of the hook curve (see upper panel in Figure 5). Dilution decreases the concentration of specific and non-specific transcripts in a parallel fashion leaving their concentration ratio virtually constant. As expected, the S/N-ratio R of selected probes remains essentially constant whereas the binding strength of specific binding progressively decreases (compare solid symbols and thick lines in the lower panel of Figure 5).

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.