In collaboration with the Center for Ecogenetics and Environmental Health (CEEH), the Microarray Facility constitutes one of two major components of CHDD's Genetics Core. Through an established agreement with the CHDD, the Microarray Facility provides a comprehensive microarray analysis resource, including the following:
- Consultation for study design and how to use the Microarray Facility
- All technical aspects associated with the wet-lab component of microarray analysis
- Complete bioinformatics and biostatistical analysis of microarray data
- Bioinformatics educational support
- Web-based access to a microarray data base and analysis tools
- Assistance in grant submission and in manuscript preparation and review
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Faculty & Staff
Federico Farin, M.D.
Theo Bammler, Ph.D.
Richard Beyer, Ph.D.
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To Use Our Services
Investigators who are interested in consulting with the Microarray Facility should:
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| Wet-Lab Services | Bioinformatics
& Biostatistics | Major Equipment
Microarray analysis is a powerful technology
that allows assessment of gene expression at a global scale suitable
to elucidate biological mechanisms. The equipment and expertise
required to carry out thorough microarray analysis are costly and
most investigators don't have the necessary resources at their disposal.
The Microarray Component of the CHDD Genetics Core provides a highly
cost effective and comprehensive microarray analysis service to
its affiliates. The specific wet-lab and bioinformatics and biostatistics
services are outlined in more detail below.
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Specific wet-lab services associated with microarray analysis include:
- Consultation on microarray platform selection (Affymetrix GeneChip® System, CodeLink Bioarray System, spotted cDNA arrays)
- Quantitative RT-PCR (reverse transcription–polymerase chain reaction) for pre-array evaluation of samples and for validation of microarray results
- An ABI 7900 Sequence Detection System allows quantitation of specific mRNA levels using fluorogenic 5' nuclease-based assays. In addition, the staff can develop novel RT-PCR assays with the dual-labeled probes positioned in close proximity to the gene location of the microarray probe.
- Total RNA extraction and RNA amplification (aRNA)
- Double amplification of very low yield RNA samples optimized for further processing with the Affymetrix GeneChip® platform
- Quality evaluation of total RNA and aRNA
- Fluorescent labeling of nucleic acids
- Microarray hybridization and scanning
- Detection of up to 100 proteins in a single sample with the Bio-Plex Suspension Array System (Bio-Rad Inc.).
- The Bio-Plex System is an “open source” fluorescent bead-based platform using the Luminex xMAP technology. Many commercial kits are available to measure a variety of proteins (e.g., cytokines, phosphoproteins involved in signal transduction, transcription factors). Among other applications, this technology can be used to validate data generated by microarray or proteomic analyses.
|The Bio-Plex Suspension Array System
enables CHDD investigators to perform rapid and sensitive immunoassays
in a high throughput, cost and time efficient manner using commercially
- Processing of high-density Affymetrix SNP (single nucleotide
polymorphism) arrays suitable for genetic association studies
as well as comparative genomic hybridization (CGH)
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Bioinformatics & Biostatistics
Transcriptional profiling generates large amounts of data. The majority of investigators do not possess the skills or expertise in bioinformatics and biostatistics to perform thorough and rigorous data analysis. In addition, research in bioinformatics and biostatistical methods for genomic data is currently developing very rapidly, and even the most statistically savvy life science investigator finds it difficult, if not impossible, to keep up with the field. The Microarray Facility addresses this issue by providing bioinformatics and biostatistics support that enables CHDD Research Affiliates to incorporate microarray technology into their research. CHDD has pooled resources with the Center for Ecogenetics and Environmental Health and the Nathan Shock Center to support Dr. Richard Beyer, a full-time bioinformaticist who has expertise in applied mathematics and extensive experience in creating and implementing mathematical algorithms.
To keep abreast of the latest developments and best practices, Dr. Beyer works in close collaboration with Dr. Kathleen Kerr in the Department of Biostatistics (consultant role) in the areas of experimental design and statistical methods. Dr. Kerr is one of several faculty members at the University of Washington and the Fred Hutchinson Cancer Research Center who have an active interest in the mathematical underpinnings of gene expression profiling using DNA microarrays. Faculty also attend national conferences and participate in advanced workshops by colleagues from other universities. Thus, CHDD affiliates have access not only to the expertise of Dr. Beyer but to the larger community of researchers at the University of Washington, the Fred Hutchinson Cancer Research Center, and nationally.
In addition, Dr. Beyer works closely with Dr. Bammler whose extensive background and experience in molecular biology provide him with the ideal background to interact with CHDD investigators concerning all biological and bioinformatic aspects associated with expression array analysis. Drs. Bammler and Beyer combine their expertise in molecular biology and applied mathematics/computer programming to form a highly effective interdisciplinary team ideally suited to meet the challenges of successful microarray bioinformatic analysis.
Specific bioinformatics and biostatistics services include the following:
- Consultation on experimental design
- General statistical consulting
- Data management and preparation (e.g., pre-processing)
- Database design and development
- Custom software design and development
- Microarray data normalization and identification of differentially expressed genes
- Microarray data visualization and exploratory analysis (e.g., clustering)
- Data analysis, interpretation and report preparation
- Assistance in grant or protocol submission (e.g., preparing statistical analysis plan or database description)
- Manuscript review and preparation
- Advanced training in microarray analysis
- Access to GeneTraffic, a microarray data management and analysis system
- Management of a bioinformatics and statistical software suite including the following items:
- Spot is a software package for the analysis of microarray images from CSIRO, Australia 's Commonwealth Scientific and Industrial Research Organisation
- CodeLink Expression Ananalysis software is used for feature extraction of tiff image files generated by GenePix software.
- MA-ANOVA software developed by the Statistical Genetics Group at the Jackson Laboratory (http://www.jax.org/research/churchill) provides tools for normalization and data transformation. The outstanding feature of MA-ANOVA is the ability to perform the analysis of variance on microarray data with methods of non-parametric inference. This allows investigators to use more sophisticated experimental designs in their microarray studies. MA-ANOVA also offers a method to incorporate error-modeling into higher order analyses such as cluster analysis.
- Bioconductor software was developed by collaborators based at the Biostatistics Unit of the Dana Farber Cancer Institute at Harvard Medical School/Harvard School of Public Health (http://bioconductor.org). The Bioconductor software provides an additional set of tools for data normalization and visualization. Bioconductor also allows for more sophisticated linear mixed-effects statistical models which can be used to describe more complex relationships between gene expression and experimental covariates.
- In the program qvalue, p-values are adjusted for multiplicity; this allows for selecting statistically significant genes while controlling the estimated “false discovery rate.”
- GeneTraffic (Iobion Informatics LLC, La Jolla, CA) is a web-accessible microarray database with a wide variety of tightly integrated analysis tools. GeneTraffic provides tools for cluster analysis and mechanisms to annotate gene lists so that investigators can begin to explore gene families and biological pathways.
- GenMAPP 2.0 has recently been released and is freely available (http://www.genmapp.org; Gladstone Institute, UCSF). GenMAPP is a useful tool in identifying biological pathways.
- PAINT (http://www.dbi.tju.edu/dbi/tools/paint/) is a software tool designed to identify common transcription factor binding sites in the 5’-untranslated regions of a list of differentially expressed genes. PAINT has been developed by the Daniel Baugh Institute at the Thomas Jefferson University and is freely available to the academic community.
- Affymetrix provides advanced software tools for acquisition, management and analysis of Affymetrix gene expression data. These include GeneChip Operating Software (GCOS) and the Data Mining Tool (DMT; will be upgraded in the near future to GeneChip RNA Expression Analysis Software, GREX). Furthermore, the web-accessible NetAffx Analysis Center provides bioinformatic tools to facilitate biological interpretation of data (http://www.affymetrix.com/index.affx).
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Six high-end computers are dedicated to microarray data analysis (a Dell OptiPlex GX 100, two Dell Precision 370s, a Dell Precision 450, and an AMD 64-bit dual core dual 275 CPU 16 GB RAM Linux workstation). The enormous amount of gene expression data generated by investigators is managed via a GeneTraffic (Iobion Informatics, La Jolla, CA) database. GeneTraffic is available on a Dell PowerEdge 2600 dual Xeon processors with 6 GB of RAM.
The following major equipment is available to process various array platforms:
- Agilent 2100 Bioanalyzer
- ABI 7900 Sequence Detection System
- Affymetrix Hybridization Oven 640
- Affymetrix GeneChip Fluidics Station 450
- Affymetrix GeneChip Scanner 3000
- Packard ScanArray 5000XL Scanner
- Axon GenePix 4000B Scanner
- Labnet 611D Incubator
- Bio-Plex Suspension Array System with an HTF Fluidics System
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