Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

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We characterized the prevalence of cockroach allergen exposure in a nationally representative sample of U.S. homes and assessed risk factors for elevated concentrations.DesignWe used data from the National Survey of Lead and Allergens in Housing, a population-based cross-sectional survey.ParticipantsParticipants were residents of 831 U.S. homes in the survey.Evaluations/MeasurementsWe analyzed allergen, questionnaire, and observational data of 831 U.S. homes.ResultsCockroach allergen (Bla g 1) concentrations exceed 2.0 U/g, a level associated with allergic sensitization, in 11% of U.S. living room floors and 13% of kitchen floors. Concentrations exceed 8.0 U/g, a level associated with asthma morbidity, in 3% of living room floors and 10% of kitchen floors. Elevated concentrations were observed in high-rise apartments, urban settings, pre-1940 constructions, and households with incomes < $20,000. Odds of having concentrations > 8.0 U/g were greatest when roach problems were reported or observed and increased with the number of cockroaches observed and with indications of recent cockroach activity.ConclusionsHousehold cockroach allergen exposure is characterized in a nationally representative context. The allergen is prevalent in many settings, at levels that may contribute to allergic sensitization and asthma morbidity.Relevance to Clinical or Professional PracticeLikelihood of exposure can be assessed by consideration of demographic and household determinants.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/ thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from theHPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.Mouse embryonic stem (ES) cells are being used with increasing frequency for constructing mutations in vitro by homologous recombination (gene targeting) that can be transferred into the mouse germ-line after blastocyst injection (1)(2)(3). Such experiments are dependent to a large extent on the availability of selectable marker genes that can be expressed in mouse ES cells. To date, two such marker genes have been used: neomycin phosphotransferase (NEO) and thymidine kinase (TK) (4-6). Cells expressing the NEO gene can be selected for by their growth in medium containing G418. Conversely, cells expressing the Herpes simplex virus TK gene can be selected against due to their death in medium containing gancyclovir. In this report, we describe the identification of sequences necessary for the expression in ES cells of a third marker gene, hypoxanthine phosphoribosyltransferase (HPRT). This gene allows two selection methods. Cells expressing the HPRT gene can be either selected for by growth in medium containing hypoxanthine/aminopterin/ thymidine (HAT) or they can be selected against by culture in medium containing 6-thioguanine (6-TG).The HPRT gene is located within a 44-kilobase (kb) region on the X chromosome in humans and rodents (7)(8)(9). It contains 9 exons, which encode a 1.3-kb mRNA. While previous studies have shown that HPRT cDNA driven by a number of different promoters can be expressed after transfection into somatic cell lines, we demonstrate in this report that similar constructs rarely yield HAT-resistant ES cell colonies. We therefore began to search for elements within the gene that influence its expression in these cells.Recent analyses of a number of genes have shown that important regulatory sequences such as transcriptional enhancers or suppressors are sometimes contained in introns (10-17). Also, several reports have suggested that the presence of an intron, even one from a foreign gene, may contribute in a general way to the level of expression of a gene...

The External RNA Control Consortium (ERCC) is an ad-hoc group with approximately 70 members from private, public, and academic organizations. The group is developing a set of external RNA control transcripts that can be used to assess technical performance in gene expression assays. The ERCC is now initiating the Testing Phase of the project, during which candidate external RNA controls will be evaluated in both microarray and QRT-PCR gene expression platforms. This document describes the proposed experiments and informatics process that will be followed to test and qualify individual controls. The ERCC is distributing this description of the proposed testing process in an effort to gain consensus and to encourage feedback from the scientific community. On October 4-5, 2005, the ERCC met to further review the document, clarify ambiguities, and plan next steps. A summary of this meeting and changes to the test plan are provided as an appendix to this manuscript.

We have investigated cotransformation in mammalian cells and its potential for identifying cells

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