Abstract
BackgroundHigh-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.ResultsThe 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).ConclusionThe 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.
Highlights
High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years
Microarray technology is a high-throughput and parallel platform that enables research on whole genomes, thereby helping to increase our understanding of the regulation of biological systems. All variations of this technique involve the deposition of a large number of probe sequences – representing a whole genome or subsets thereof – in a regular grid-like array on a physical substrate, usually a glass slide for custom spotted arrays
As opposed to high-density arrays like Affymetrix, standard experiments using spotted arrays on glass slides usually involve the co-hybridisation of two differently labelled targets to one slide. This is normally referred to as dual-target or dual-dye arrays. In such a cDNA microarray dual-target experiment, it is usually the fluorescent dyes Cy5 and Cy3 that are used in tandem
Summary
High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. Microarray technology is a high-throughput and parallel platform that enables research on whole genomes, thereby helping to increase our understanding of the regulation of biological systems All variations of this technique involve the deposition of a large number of probe sequences (e.g. oligonucleotides, cDNA) – representing a whole genome or subsets thereof – in a regular grid-like array on a physical substrate, usually a glass slide for custom spotted arrays. As opposed to high-density arrays like Affymetrix (probes produced in-situ in a process resembling lithography), standard experiments using spotted arrays on glass slides usually involve the co-hybridisation of two differently labelled targets to one slide This is normally referred to as dual-target or dual-dye arrays. Location normalisation methods like LoWeSS are used to (non-linearly) normalise log ratios within an array, additional methods for normalisation of scale are used to compare log-ratios across multiple arrays
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