Summaries of Affymetrix GeneChip probe level data.

High density oligonucleotide array technology is widely used in many areas of biomedical research for quantitative and highly parallel measurements of gene expression. Affymetrix GeneChip arrays are the most popular. In this technology each gene is typically represented by a set of 11-20 pairs of probes. In order to obtain expression measures it is necessary to summarize the probe level data. Using two extensive spike-in studies and a dilution study, we developed a set of tools for assessing the effectiveness of expression measures. We found that the performance of the current version of the default expression measure provided by Affymetrix Microarray Suite can be significantly improved by the use of probe level summaries derived from empirically motivated statistical models. In particular, improvements in the ability to detect differentially expressed genes are demonstrated.

Argon laser photocoagulation-induced modification of gene expression in the retina.

PURPOSE: To generate a profile of genes expressed in the retina, RPE, and choroid after laser treatment and to identify genes that may contribute to the beneficial effects of laser photocoagulation in the treatment of angiogenic retinal diseases. METHODS: Argon laser irradiation was delivered to the left eye of normal C57BL/6J mice (n = 30), with the right eye serving as the control in each animal. Three days after laser treatment, mice were culled, eyes enucleated, and the retinas dissected and pooled into respective groups. The total RNA of replicate samples was extracted, and expression profiles were obtained by microarray analysis. Data comparisons between control and treated samples were performed and statistically analyzed. RESULTS: Data revealed that the expression of 265 known genes and expressed sequence tags (ESTs) changed after laser treatment. Of those, 25 were found to be upregulated. These genes represented a number of biological processes, including photoreceptor metabolism, synaptic function, structural proteins, and adhesion molecules. Thus angiotensin II type 2 receptor (Agtr2), a potential candidate in the inhibition of VEGF-induced angiogenesis, was upregulated, whereas potential modulators of endothelial cell function, permeability factors, and VEGF inducers, such as FGF-14, FGF-16, IL-1beta, calcitonin receptor-like receptor (CRLR), and plasminogen activator inhibitor-2 (PAI2), were downregulated. CONCLUSIONS: In this study, genes were identified that both explain and contribute to the beneficial effects of laser photocoagulation in the treatment of angiogenic retinal diseases. The molecular insights into the therapeutic effects of laser photocoagulation may provide a basis for future therapeutic strategies.

The microarray: potential applications for ophthalmic research.

The microarray is a revolutionary technology combining molecular biology and computer technology in the high throughput, simultaneous analysis of global gene expression. It is emerging as a powerful and valuable research tool that holds great promise in elucidating the molecular mechanisms involved in complex diseases. The information gained may provide direction toward identifying appropriate targets for therapeutic intervention. Despite the enormous potential of this technology, however, a number of issues exist that complicate gene expression analysis and require further resolution. This paper reviews these issues as well as the conceptual, practical and statistical aspects of microarray technology, including its current use in research and clinical applications. Furthermore, the advantages and potential benefits of this technology in ophthalmic research are discussed, with particular attention to retinal diseases, and its possible application in the identification of genes involved in ocular disease progression that may serve as clinical markers or potential therapeutic targets.

Local recurrence map to guide target volume delineation after radical prostatectomy.

PURPOSE: To describe the various anatomic locations of recurrent disease, in a cohort of men with radiographically visualized, biopsy-proven recurrent prostate cancer after radical prostatectomy (RP), in order to help guide contouring of the prostatic fossa clinical target volume (PF-CTV) when no gross recurrence is visible or when magnetic resonance imaging (MRI) is not used. METHODS AND MATERIALS: Ten representative patients with MRI-detected, biopsy-proven local recurrences of prostate adenocarcinoma after RP were selected. Areas of recurrence were delineated on individual MRI images, and then mapped onto axial and sagittal "template" MRI images to compositely demonstrate the documented areas of recurrence. Coverage of these anatomic areas of recurrence was then evaluated by applying Radiation Therapy Oncology Group (RTOG)-consensus PF-CTV contours to a postoperative computed tomographic template. RESULTS: The median age at the time of RP was 61 years (range, 50-73). In the superoinferior direction, recurrences ranged from the superior retrovesical region, to the inferior retrovesical region, to the posterior anastomosis, and as inferiorly as the posterior urogenital diaphragm. In the anteroposterior direction, the areas of recurrence ranged from involving the posterior bladder wall anteriorly to invading the rectum posteriorly. Recurrences were found at the center, right, and left of the prostate and seminal vesicle fossa. When target volumes were delineated using RTOG-defined consensus PF-CTV contours, coverage was marginal on recurrences in the posterolateral aspects of the CTV near the rectum and mesorectal fascia and lacking on recurrences occurring inferiorly at the posterior urogenital diaphragm. CONCLUSIONS: Our findings describe the variation in location of prostate cancer recurrences and can be used to improve target definition in conformal radiation therapy in the postoperative adjuvant or salvage setting. RTOG-consensus contours for the PF-CTV should be applied carefully, with potential modifications in the posterolateral and inferior aspects.

Exploration, normalization, and summaries of high density oligonucleotide array probe level data.

In this paper we report exploratory analyses of high-density oligonucleotide array data from the Affymetrix GeneChip system with the objective of improving upon currently used measures of gene expression. Our analyses make use of three data sets: a small experimental study consisting of five MGU74A mouse GeneChip arrays, part of the data from an extensive spike-in study conducted by Gene Logic and Wyeth's Genetics Institute involving 95 HG-U95A human GeneChip arrays; and part of a dilution study conducted by Gene Logic involving 75 HG-U95A GeneChip arrays. We display some familiar features of the perfect match and mismatch probe (PM and MM) values of these data, and examine the variance-mean relationship with probe-level data from probes believed to be defective, and so delivering noise only. We explain why we need to normalize the arrays to one another using probe level intensities. We then examine the behavior of the PM and MM using spike-in data and assess three commonly used summary measures: Affymetrix's (i) average difference (AvDiff) and (ii) MAS 5.0 signal, and (iii) the Li and Wong multiplicative model-based expression index (MBEI). The exploratory data analyses of the probe level data motivate a new summary measure that is a robust multi-array average (RMA) of background-adjusted, normalized, and log-transformed PM values. We evaluate the four expression summary measures using the dilution study data, assessing their behavior in terms of bias, variance and (for MBEI and RMA) model fit. Finally, we evaluate the algorithms in terms of their ability to detect known levels of differential expression using the spike-in data. We conclude that there is no obvious downside to using RMA and attaching a standard error (SE) to this quantity using a linear model which removes probe-specific affinities.