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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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Triple-target: data before normalisation Each panel shows the data distribution for 5 individual samples (one per array) for a particular dye-label. Array names are numbered 1–5. Samples labelled with Cy5 have a wider distribution and generally lower centre than the other two channels, with high consistency across the 5 replicate arrays.
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Figure 1: Triple-target: data before normalisation Each panel shows the data distribution for 5 individual samples (one per array) for a particular dye-label. Array names are numbered 1–5. Samples labelled with Cy5 have a wider distribution and generally lower centre than the other two channels, with high consistency across the 5 replicate arrays.

Mentions: A first investigation of the expression values obtained from the three individual samples on an array (Fig. 1, Table 1) shows that, across all probes on an array, Cy3 and Alexa594 share similar average expression and spread of values before applying any normalisation methods. Cy5 labelled samples appear to have a greater spread of data values, with differences apparent in the lower signal intensities. This may be caused by dye-label incorporation differences, which are known to occur in most common Cy5/Cy3 dual-target experiments. These differences are not evident for Cy3/Alexa594 combinations here. Before normalisation, log-ratios for pair-wise sample comparisons on the arrays are therefore showing slightly greater variance for those combinations that involve Cy5 (Fig. 2a), in addition to global differences that systematically move the ratios away from zero 0. Subsequent location and scale normalisation reduces these systematic differences and results in very comparable data distributions, all gene probes on an array contained in the Inter-Quartile-Range having log-ratios within the interval [+0.25; -0.25] (Fig. 2b).


Triple-target microarray experiments: a novel experimental strategy.

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

Triple-target: data before normalisation Each panel shows the data distribution for 5 individual samples (one per array) for a particular dye-label. Array names are numbered 1–5. Samples labelled with Cy5 have a wider distribution and generally lower centre than the other two channels, with high consistency across the 5 replicate arrays.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Triple-target: data before normalisation Each panel shows the data distribution for 5 individual samples (one per array) for a particular dye-label. Array names are numbered 1–5. Samples labelled with Cy5 have a wider distribution and generally lower centre than the other two channels, with high consistency across the 5 replicate arrays.
Mentions: A first investigation of the expression values obtained from the three individual samples on an array (Fig. 1, Table 1) shows that, across all probes on an array, Cy3 and Alexa594 share similar average expression and spread of values before applying any normalisation methods. Cy5 labelled samples appear to have a greater spread of data values, with differences apparent in the lower signal intensities. This may be caused by dye-label incorporation differences, which are known to occur in most common Cy5/Cy3 dual-target experiments. These differences are not evident for Cy3/Alexa594 combinations here. Before normalisation, log-ratios for pair-wise sample comparisons on the arrays are therefore showing slightly greater variance for those combinations that involve Cy5 (Fig. 2a), in addition to global differences that systematically move the ratios away from zero 0. Subsequent location and scale normalisation reduces these systematic differences and results in very comparable data distributions, all gene probes on an array contained in the Inter-Quartile-Range having log-ratios within the interval [+0.25; -0.25] (Fig. 2b).

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