How molecular profiling could revolutionize drug discovery.

Information from genomic, proteomic and metabolomic measurements has already benefited target discovery and validation, assessment of efficacy and toxicity of compounds, identification of disease subgroups and the prediction of responses of individual patients. Greater benefits can be expected from the application of these technologies on a significantly larger scale; by simultaneously collecting diverse measurements from the same subjects or cell cultures; by exploiting the steadily improving quantitative accuracy of the technologies; and by interpreting the emerging data in the context of underlying biological models of increasing sophistication. The benefits of applying molecular profiling to drug discovery and development will include much lower failure rates at all stages of the drug development pipeline, faster progression from discovery through to clinical trials and more successful therapies for patient subgroups. Upheavals in existing organizational structures in the current 'conveyor belt' models of drug discovery might be required to take full advantage of these methods.

How molecular profiling is transforming drug discovery.

Extract: We now have the technical ability to simultaneously determine the activities of most of the genes in the genome in a particular cell type under a particular drug, genetic, disease, or environmental condition. This ability is rapidly changing the way new targets for therapeutic intervention are chosen, how candidate drugs are prioritized in development for probable toxicity and efficacy, and how patient subgroups are matched with the best treatment options. Diagnostic tests based on these technologies will soon enter clinical practice. More detailed description on this subject was published elsewhere. "Molecular profiling," as considered here, is any combination or individual application of technologies for simultaneously measuring a large number of gene products such as messenger RNAs, proteins, or metabolites. A premier example is mRNA expression profiling with DNA microarrays. Highly parallel protein measurements based on both mass spectrometry and on microarrays of antibodies also are maturing rapidly. Scans of genetic variation among individuals of a given species also can be done encompassing thousands of genome locations.

Applications of DNA microarrays in biology.

DNA microarrays have enabled biology researchers to conduct large-scale quantitative experiments. This capacity has produced qualitative changes in the breadth of hypotheses that can be explored. In what has become the dominant mode of use, changes in the transcription rate of nearly all the genes in a genome, taking place in a particular tissue or cell type, can be measured in disease states, during development, and in response to intentional experimental perturbations, such as gene disruptions and drug treatments. The response patterns have helped illuminate mechanisms of disease and identify disease subphenotypes, predict disease progression, assign function to previously unannotated genes, group genes into functional pathways, and predict activities of new compounds. Directed at the genome sequence itself, microarrays have been used to identify novel genes, binding sites of transcription factors, changes in DNA copy number, and variations from a baseline sequence, such as in emerging strains of pathogens or complex mutations in disease-causing human genes. They also serve as a general demultiplexing tool to sort spatially the sequence-tagged products of highly parallel reactions performed in solution. A brief review of microarray platform technology options, and of the process steps involved in complete experiment workflows, is included.

Microarray standard data set and figures of merit for comparing data processing methods and experiment designs.

MOTIVATION: There is a very large and growing level of effort toward improving the platforms, experiment designs, and data analysis methods for microarray expression profiling. Along with a growing richness in the approaches there is a growing confusion among most scientists as to how to make objective comparisons and choices between them for different applications. There is a need for a standard framework for the microarray community to compare and improve analytical and statistical methods. RESULTS: We report on a microarray data set comprising 204 in-situ synthesized oligonucleotide arrays, each hybridized with two-color cDNA samples derived from 20 different human tissues and cell lines. Design of the approximately 24 000 60mer oligonucleotides that report approximately 2500 known genes on the arrays, and design of the hybridization experiments, were carried out in a way that supports the performance assessment of alternative data processing approaches and of alternative experiment and array designs. We also propose standard figures of merit for success in detecting individual differential expression changes or expression levels, and for detecting similarities and differences in expression patterns across genes and experiments. We expect this data set and the proposed figures of merit will provide a standard framework for much of the microarray community to compare and improve many analytical and statistical methods relevant to microarray data analysis, including image processing, normalization, error modeling, combining of multiple reporters per gene, use of replicate experiments, and sample referencing schemes in measurements based on expression change. AVAILABILITY/SUPPLEMENTARY INFORMATION: Expression data and supplementary information are available at http://www.rii.com/publications/2003/HE_SDS.htm

Effects of atmospheric ozone on microarray data quality.

A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect. Controlled exposures of microarrays to ozone confirmed this factor as the root cause, and we present data that show susceptibility of a class of cyanine dyes (e.g., Cy5, Alexa 647) to ozone levels as low as 5-10 ppb for periods as short as 10-30 s. Other cyanine dyes (e.g., Cy3, Alexa 555) were not significantly affected until higher ozone levels (> 100 ppb). To address this environmental effect, laboratory ozone levels should be kept below 2 ppb (e.g., with filters in HVAC) to achieve high quality microarray data.

Genetics of gene expression surveyed in maize, mouse and man.

Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma. One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression. Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes. Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes. Furthermore, we find that these obesity subtypes are under the control of different loci.

Integrated genomic and proteomic analyses of gene expression in Mammalian cells.

Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs.