Learning from microarray interlaboratory studies: measures of precision for gene expression

<p>Abstract</p> <p>Background</p> <p>The ability to demonstrate the reproducibility of gene expression microarray results is a critical consideration for the use of microarray technology in clinical applications. While studies have asserted that microarray data can be "highly reproducible" under given conditions, there is little ability to quantitatively compare amongst the various metrics and terminology used to characterize and express measurement performance. Use of standardized conceptual tools can greatly facilitate communication among the user, developer, and regulator stakeholders of the microarray community. While shaped by less highly multiplexed systems, measurement science (metrology) is devoted to establishing a coherent and internationally recognized vocabulary and quantitative practice for the characterization of measurement processes.</p> <p>Results</p> <p>The two independent aspects of the metrological concept of "accuracy" are "trueness" (closeness of a measurement to an accepted reference value) and "precision" (the closeness of measurement results to each other). A carefully designed collaborative study enables estimation of a variety of gene expression measurement precision metrics: repeatability, several flavors of intermediate precision, and reproducibility. The three 2004 Expression Analysis Pilot Proficiency Test collaborative studies, each with 13 to 16 participants, provide triplicate microarray measurements on each of two reference RNA pools. Using and modestly extending the consensus ISO 5725 documentary standard, we evaluate the metrological precision figures of merit for individual microarray signal measurement, building from calculations appropriate to single measurement processes, such as technical replicate expression values for individual probes on a microarray, to the estimation and display of precision functions representing all of the probes in a given platform.</p> <p>Conclusion</p> <p>With only modest extensions, the established metrological framework can be fruitfully used to characterize the measurement performance of microarray and other highly multiplexed systems. Precision functions, summarizing routine precision metrics estimated from appropriately repeated measurements of one or more reference materials as functions of signal level, are demonstrated and merit further development for characterizing measurement platforms, monitoring changes in measurement system performance, and comparing performance among laboratories or analysts.</p

Research proposal on certain atomic physics measurements associated with the laser isotope separation method

Research proposed in the following areas are briefly described: the measurement of photoionization cross sections near threshold for the excited states of uranium; measurement of the symmetric charge exchange cross section; and measurement of chemiionization cross sections for electronically excited uranium atoms

Xray computed tomography in Zernike phase contrast mode at 8 keV with 50-nm resolution using Cu rotating anode x-ray source

Abstract. High-resolution X-ray computed tomography (XCT) enables nondestructive 3D imaging of complex structures, regardless of their state of crystallinity. This work describes a sub-50 nm resolution XCT system operating at 8 keV in absorption and Zernike phase contrast modes based on a commercially available Cu rotating anode laboratory X-ray source. The system utilizes a high efficiency reflective capillary condenser lens and high-resolution Fresnel zone plates with an outermost zone width of 35 nm and 700 nm structure height resulting in a spatial resolution better than 50 nm currently. Imaging a fragment of the solid oxide fuel cells (SOFC) with 50 nm resolution is presented as an application example of the XCT technique in materials science and nanotechnology

Non Destructive Failure Analysis Technique With a Laboratory Based 3D X-ray Nanotomography System

ABSTRACT X-ray computed tomography (CT) is a powerful nondestructive 3D imaging technique, which enables the visualization of the three dimensional internal structure of opaque materials such as semiconductor devices. Reports of high resolution CT research on life science, materials and semiconductor has mainly been confined to synchrotron radiation centers. This severely limits the availability and accessibility of x-ray microscopes and the wide proliferation of this methodology. We describe a sub-50nm resolution nanoCT system operating at 8 keV in Zernike phase contrast mode based on a commercially available laboratory x-ray source. The system utilizes high-efficiency Fresnel zone plates with an outermost zone width of 35nm resulting in spatial resolution better than 50 nm. The technical description of the system and failure analysis applications notably in visualizing voids, residues in metal interconnects, and competitive analysis in semiconductor devices will be discussed

Exploring the use of internal and externalcontrols for assessing microarray technical performance

<p>Abstract</p> <p>Background</p> <p>The maturing of gene expression microarray technology and interest in the use of microarray-based applications for clinical and diagnostic applications calls for quantitative measures of quality. This manuscript presents a retrospective study characterizing several approaches to assess technical performance of microarray data measured on the Affymetrix GeneChip platform, including whole-array metrics and information from a standard mixture of external spike-in and endogenous internal controls. Spike-in controls were found to carry the same information about technical performance as whole-array metrics and endogenous "housekeeping" genes. These results support the use of spike-in controls as general tools for performance assessment across time, experimenters and array batches, suggesting that they have potential for comparison of microarray data generated across species using different technologies.</p> <p>Results</p> <p>A layered PCA modeling methodology that uses data from a number of classes of controls (spike-in hybridization, spike-in polyA+, internal RNA degradation, endogenous or "housekeeping genes") was used for the assessment of microarray data quality. The controls provide information on multiple stages of the experimental protocol (e.g., hybridization, RNA amplification). External spike-in, hybridization and RNA labeling controls provide information related to both assay and hybridization performance whereas internal endogenous controls provide quality information on the biological sample. We find that the variance of the data generated from the external and internal controls carries critical information about technical performance; the PCA dissection of this variance is consistent with whole-array quality assessment based on a number of quality assurance/quality control (QA/QC) metrics.</p> <p>Conclusions</p> <p>These results provide support for the use of both external and internal RNA control data to assess the technical quality of microarray experiments. The observed consistency amongst the information carried by internal and external controls and whole-array quality measures offers promise for rationally-designed control standards for routine performance monitoring of multiplexed measurement platforms.</p

Robust Detection of Hierarchical Communities from Escherichia coli Gene Expression Data

Determining the functional structure of biological networks is a central goal of systems biology. One approach is to analyze gene expression data to infer a network of gene interactions on the basis of their correlated responses to environmental and genetic perturbations. The inferred network can then be analyzed to identify functional communities. However, commonly used algorithms can yield unreliable results due to experimental noise, algorithmic stochasticity, and the influence of arbitrarily chosen parameter values. Furthermore, the results obtained typically provide only a simplistic view of the network partitioned into disjoint communities and provide no information of the relationship between communities. Here, we present methods to robustly detect coregulated and functionally enriched gene communities and demonstrate their application and validity for Escherichia coli gene expression data. Applying a recently developed community detection algorithm to the network of interactions identified with the context likelihood of relatedness (CLR) method, we show that a hierarchy of network communities can be identified. These communities significantly enrich for gene ontology (GO) terms, consistent with them representing biologically meaningful groups. Further, analysis of the most significantly enriched communities identified several candidate new regulatory interactions. The robustness of our methods is demonstrated by showing that a core set of functional communities is reliably found when artificial noise, modeling experimental noise, is added to the data. We find that noise mainly acts conservatively, increasing the relatedness required for a network link to be reliably assigned and decreasing the size of the core communities, rather than causing association of genes into new communities.Comment: Due to appear in PLoS Computational Biology. Supplementary Figure S1 was not uploaded but is available by contacting the author. 27 pages, 5 figures, 15 supplementary file

NIST interlaboratory study on glycosylation analysis of monoclonal antibodies : comparison of results from diverse analytical methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy‑six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation  analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type.. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods

Efeito de reguladores de crescimento sobre os teores de ácido ascórbico e carboidratos solúveis de morango (Fragaria hybridus)

Several growth regulators were sprayed on strawberry plants: SADH (5000 ppm), CCC (2000 ppm), IAA (10 ppm, 3 times), GA 10 ppm, 3 times) and GA (550 ppm), Ascorbic acid, dry weight and soluble carbohydrates contents of fruits were determined. Statistically differences were not observed between treatments and control. Dry weight varied from 7.62 to 9.53%. Ascorbic acid content varied from 35.88 to 71.81 mg/100 g on fresh weight basis. Mean values of soluble carbohydrates, in grams/100 g on fresh weight basis, were total (5.58), sucrose (1.01), glucose (1.63) and frutose (1.54).Foram feitas aplicações dos seguintes reguladores de crescimento em morango (Fragarm hybridus): SADH a 5000 ppm, CCC a 2000 ppm, ATA a 10 ppm (3 aplicações), ácido giberélico a 10 ppm (3 aplicações) e 550 ppm. Análises de peso seco, ácido ascórbico e carboidratos solúveis dos frutos foram efetuadas a fim de se estudar o efeito desses reguladores de crescimento. Não foram detectadas diferenças significativas entre os diversas tratamentos e o controle. O peso seco variou de 7,62 a 9,53%. O conteúdo de ácido ascórbico variou de 35,88 a 71,81 mg/100 g de peso fresco. Os teores médios de carboidratos solúveis, expresso em g/100 g peso fresco, foram: totais (5,58), sacarose (1,01), glucose (1,63) e frutose (1,54)