Experiments using microarray technology: limitations and standard operating procedures

Microarrays are a powerful method for the global analysis of gene or protein content and expression, opening up new horizons in molecular and physiological systems. This review focuses on the critical aspects of acquiring meaningful data for analysis following fluorescence-based target hybridisation to arrays. Although microarray technology is adaptable to the analysis of a range of biomolecules (DNA, RNA, protein, carbohydrates and lipids), the scheme presented here is applicable primarily to customised DNA arrays fabricated using long oligomer or cDNA probes. Rather than provide a comprehensive review of microarray technology and analysis techniques, both of which are large and complex areas, the aim of this paper is to provide a restricted overview, highlighting salient features to provide initial guidance in terms of pitfalls in planning and executing array projects. We outline standard operating procedures, which help streamline the analysis of microarray data resulting from a diversity of array formats and biological systems. We hope that this overview will provide practical initial guidance for those embarking on microarray studies

Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon-γ in primary macrophages requires endogenous 25-hydroxycholesterol synthesis (in press)

AbstractInterferons (IFNs) play a central role in immunity and emerging evidence suggests that IFN-signalling coordinately regulates sterol biosynthesis in macrophages, via Sterol Regulatory Element-Binding Protein (SREBP) dependent and independent pathways. However, the precise mechanisms and kinetic steps by which IFN controls sterol biosynthesis are as yet not fully understood. Here, we elucidate the molecular circuitry governing how IFN controls the first regulated step in the mevalonate-sterol pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), through the synthesis of 25-Hydroxycholesterol (25-HC) from cholesterol by the IFN-inducible Cholesterol-25-Hydroxylase (CH25H). We show for the first 30-min of IFN stimulation of macrophages the rate of de novo synthesis of the Ch25h transcript is markedly increased but by 120-min becomes transcriptionally curtailed, coincident with induction of the Activating Transcription Factor 3 (ATF3) repressor. We demonstrate ATF3 induction by Toll-like receptors is strictly dependent on IFN-signalling. While the SREBP-pathway dependent rates of de novo transcription of Hmgcr are relatively unchanged in the first 90-min of IFN treatment, we find HMGCR enzyme levels undergo a rapid proteasomal-mediated degradation, defining a previously unappreciated SREBP-independent mechanism for IFN-action. These events precede a sustained marked reduction in Hmgcr RNA levels involving SREBP-dependent mechanisms. We demonstrate that HMGCR proteasomal-degradation by IFN strictly requires the synthesis of endogenous 25-HC and functionally couples HMGCR to CH25H to coordinately suppress sterol biosynthesis. In conclusion, we quantitatively delineate proteomic and transcriptional levels of IFN-mediated control of HMGCR, the primary enzymatic step of the mevalonate-sterol biosynthesis pathway, providing a foundational framework for mathematically modelling the therapeutic outcome of immune-metabolic pathways

Triple-target microarray experiments: a novel experimental strategy

<p>Abstract</p> <p>Background</p> <p>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 <it>three </it>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.</p> <p>Results</p> <p>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).</p> <p>Conclusion</p> <p>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.</p

Transcriptomal analysis of varicella-zoster virus infection using long oligonucleotide-based microarrays

Varicella-zoster virus (VZV) is a human herpes virus that causes varicella as a primary infection and herpes zoster following reactivation of the virus from a latent state in trigeminal and spinal ganglia. In order to study the global pattern of VZV gene transcription, VZV microarrays using 75-base oligomers to 71 VZV open reading frames (ORFs) were designed and validated. The long-oligonucleotide approach maximizes the stringency of detection and polarity of gene expression. To optimize sensitivity, microarrays were hybridized to target RNA and the extent of hybridization measured using resonance light scattering. Microarray data were normalized to a subset of invariant ranked host-encoded positive-control genes and the data subjected to robust formal statistical analysis. The programme of viral gene expression was determined for VZV (Dumas strain)-infected MeWo cells and SVG cells (an immortalized human astrocyte cell line) 72 h post-infection. Marked quantitative and qualitative differences in the viral transcriptome were observed between the two different cell types using the Dumas laboratory-adapted strain. Oligonucleotide-based VZV arrays have considerable promise as a valuable tool in the analysis of viral gene transcription during both lytic and latent infections, and the observed heterogeneity in the global pattern of viral gene transcription may also have diagnostic potentia