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Triple-target microarray experiments: a novel experimental strategy.

Forster T, Costa Y, Roy D, Cooke HJ, Maratou K - BMC Genomics (2004)

Bottom Line: We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design.These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design.In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scottish Centre for Genomic Technology and Informatics, University of Edinburgh, The Chancellor's Building, College of Medicine, 49 Little France Crescent, Edinburgh, EH16 4SB, UK. Thorsten.Forster@ed.ac.uk

ABSTRACT

Background: High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. There are currently two main dye-labelling strategies for microarray studies based on custom-spotted cDNA or oligonucleotides arrays: (I) Dye-labelling of a single target sample with a particular dye, followed by subsequent hybridisation to a single microarray slide, (II) Dye-labelling of two different target samples with two different dyes, followed by subsequent co-hybridisation to a single microarray slide. The two dyes most frequently used for either method are Cy3 and Cy5. We propose and evaluate a novel experiment set-up utilising three differently labelled targets co-hybridised to one microarray slide. In addition to Cy3 and Cy5, this incorporates Alexa 594 as a third dye-label. We evaluate this approach in line with current data processing and analysis techniques for microarrays, and run separate analyses on Alexa 594 used in single-target, dual-target and the intended triple-target experiment set-ups (a total of 18 microarray slides). We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design.

Results: The addition of Alexa 594 as a dye-label for an additional--third--target sample works within the framework of more commonplace Cy5/Cy3 labelled target sample combinations. Standard normalisation methods are still applicable, and the resulting data can be expected to allow identification of expression differences in a biological experiment, given sufficient levels of biological replication (as is necessary for most microarray experiments).

Conclusion: The use of three dye-labelled target samples can be a valuable addition to the standard repertoire of microarray experiment designs. The method enables direct comparison between two experimental populations as well as measuring these two populations in relation to a third reference sample, allowing comparisons within the slide and across slides. These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design. Common methods for data processing and analysis are still applicable, but there is scope for the development of custom models for triple-target data. In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

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Dual-target: data before normalisation For all dual-target hybridisations, these box-plots show the signal distributions across all genes for each individual dye-label sample. The non-hybridised "blank" dye-label channel has also been scanned and processed, in order to assess the effect of signal bleeding from one dye-label to another. The only truly blank scan is Cy5 for the Cy3/Alexa594 co-hybridisations. Co-hybridisations of the standard Cy5/Cy3 and new Cy5/Alexa594 dye-labels leads to small, but detectable levels of signal in the blank scan. Regarding spread of data, the dual-target hybridisation are identical to the results of the triple-target hybridisations, with a wider distribution and lower average of signal for Cy5.
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Figure 6: Dual-target: data before normalisation For all dual-target hybridisations, these box-plots show the signal distributions across all genes for each individual dye-label sample. The non-hybridised "blank" dye-label channel has also been scanned and processed, in order to assess the effect of signal bleeding from one dye-label to another. The only truly blank scan is Cy5 for the Cy3/Alexa594 co-hybridisations. Co-hybridisations of the standard Cy5/Cy3 and new Cy5/Alexa594 dye-labels leads to small, but detectable levels of signal in the blank scan. Regarding spread of data, the dual-target hybridisation are identical to the results of the triple-target hybridisations, with a wider distribution and lower average of signal for Cy5.

Mentions: All arrays confirm the results obtained from analysis I: Cy5 is associated with more gene probes values in the low expression range (Fig. 6). The dependence of log-ratio on log-intensity of a gene probe is also consistent with the triple-target arrays, in that the use of Cy5 with either of the other two dyes leads to non-linear effects (Fig. 7). A positive aspect of Cy5 are the very small signal values close to zero when it is the blank channel, which cannot be said for Alexa594 and Cy3, both of which result in low level signal values even if no sample has been hybridised to the array with the corresponding dye-label. This is probably an indication of the relative closeness of the dye-labels in the light frequency spectrum, leading for example to fluorescence of the Cy3 channel when the array is subjected with the laser frequency corresponding to Alexa594. However, the level of signal obtained from these blank channels is small in proportion to the hybridised channels, with linear slopes between 0.02 and 0.03 and intercept signal values between 52 and 115. Given the assumption that this effect is also present in the triple-target hybridisation, it does not present itself as a large or non-systematic problem. It does not cause signal interpretation problems that are greater than those created by using two dyes with a non-linear relationship (i.e. Cy5 vs. Cy3) at comparable low levels of expression.


Triple-target microarray experiments: a novel experimental strategy.

Forster T, Costa Y, Roy D, Cooke HJ, Maratou K - BMC Genomics (2004)

Dual-target: data before normalisation For all dual-target hybridisations, these box-plots show the signal distributions across all genes for each individual dye-label sample. The non-hybridised "blank" dye-label channel has also been scanned and processed, in order to assess the effect of signal bleeding from one dye-label to another. The only truly blank scan is Cy5 for the Cy3/Alexa594 co-hybridisations. Co-hybridisations of the standard Cy5/Cy3 and new Cy5/Alexa594 dye-labels leads to small, but detectable levels of signal in the blank scan. Regarding spread of data, the dual-target hybridisation are identical to the results of the triple-target hybridisations, with a wider distribution and lower average of signal for Cy5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC365026&req=5

Figure 6: Dual-target: data before normalisation For all dual-target hybridisations, these box-plots show the signal distributions across all genes for each individual dye-label sample. The non-hybridised "blank" dye-label channel has also been scanned and processed, in order to assess the effect of signal bleeding from one dye-label to another. The only truly blank scan is Cy5 for the Cy3/Alexa594 co-hybridisations. Co-hybridisations of the standard Cy5/Cy3 and new Cy5/Alexa594 dye-labels leads to small, but detectable levels of signal in the blank scan. Regarding spread of data, the dual-target hybridisation are identical to the results of the triple-target hybridisations, with a wider distribution and lower average of signal for Cy5.
Mentions: All arrays confirm the results obtained from analysis I: Cy5 is associated with more gene probes values in the low expression range (Fig. 6). The dependence of log-ratio on log-intensity of a gene probe is also consistent with the triple-target arrays, in that the use of Cy5 with either of the other two dyes leads to non-linear effects (Fig. 7). A positive aspect of Cy5 are the very small signal values close to zero when it is the blank channel, which cannot be said for Alexa594 and Cy3, both of which result in low level signal values even if no sample has been hybridised to the array with the corresponding dye-label. This is probably an indication of the relative closeness of the dye-labels in the light frequency spectrum, leading for example to fluorescence of the Cy3 channel when the array is subjected with the laser frequency corresponding to Alexa594. However, the level of signal obtained from these blank channels is small in proportion to the hybridised channels, with linear slopes between 0.02 and 0.03 and intercept signal values between 52 and 115. Given the assumption that this effect is also present in the triple-target hybridisation, it does not present itself as a large or non-systematic problem. It does not cause signal interpretation problems that are greater than those created by using two dyes with a non-linear relationship (i.e. Cy5 vs. Cy3) at comparable low levels of expression.

Bottom Line: We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design.These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design.In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scottish Centre for Genomic Technology and Informatics, University of Edinburgh, The Chancellor's Building, College of Medicine, 49 Little France Crescent, Edinburgh, EH16 4SB, UK. Thorsten.Forster@ed.ac.uk

ABSTRACT

Background: High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. There are currently two main dye-labelling strategies for microarray studies based on custom-spotted cDNA or oligonucleotides arrays: (I) Dye-labelling of a single target sample with a particular dye, followed by subsequent hybridisation to a single microarray slide, (II) Dye-labelling of two different target samples with two different dyes, followed by subsequent co-hybridisation to a single microarray slide. The two dyes most frequently used for either method are Cy3 and Cy5. We propose and evaluate a novel experiment set-up utilising three differently labelled targets co-hybridised to one microarray slide. In addition to Cy3 and Cy5, this incorporates Alexa 594 as a third dye-label. We evaluate this approach in line with current data processing and analysis techniques for microarrays, and run separate analyses on Alexa 594 used in single-target, dual-target and the intended triple-target experiment set-ups (a total of 18 microarray slides). We follow this by pointing out practical applications and suitable analysis methods, and conclude that triple-target microarray experiments can add value to microarray research by reducing material costs for arrays and related processes, and by increasing the number of options for pragmatic experiment design.

Results: The addition of Alexa 594 as a dye-label for an additional--third--target sample works within the framework of more commonplace Cy5/Cy3 labelled target sample combinations. Standard normalisation methods are still applicable, and the resulting data can be expected to allow identification of expression differences in a biological experiment, given sufficient levels of biological replication (as is necessary for most microarray experiments).

Conclusion: The use of three dye-labelled target samples can be a valuable addition to the standard repertoire of microarray experiment designs. The method enables direct comparison between two experimental populations as well as measuring these two populations in relation to a third reference sample, allowing comparisons within the slide and across slides. These benefits are only offset by the added level of consideration required in the experimental design and data processing of a triple-target study design. Common methods for data processing and analysis are still applicable, but there is scope for the development of custom models for triple-target data. In summary, we do not consider the triple-target approach to be a new standard, but a valuable addition to the existing microarray study toolkit.

Show MeSH
Related in: MedlinePlus