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to the DERC Website: Genomics Core
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Genomics Core |
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Location: Room 607, Joslin Diabetes Center
Phone: 617-264-2795, internal ext. 4543
Research Technician: Josh Schroeder, Joshua.Schroeder@joslin.harvard.edu
Core Director: Mary Elizabeth Patti, Mary.Elizabeth.Patti@joslin.harvard.edu
***The Genomics Core now supports Affymetrix 2.0 arrays***
 | The Joslin Genomics Core is a core facility for the NIH-sponsored Diabetes Genome Anatomy Project (DGAP) - please visit: www.diabetesgenome.org |
The new PCR Core is part of the Genomics Core.
Methodology summary:
High-density oligonucleotide arrays allow simultaneous and quantitative analysis of expression of thousands of genes and EST and provide an efficient way to determine the full pattern of alterations in gene expression present in a wide variety of experimental conditions and pathologic states. Affymetrix chips are high-density collections of 25-mer oligonucleotides aligned in arrays on a silicon chip. Each gene or EST is represented on the array by 20 probe pairs, consisting of a perfect match (PM) and a mismatch (MM) oligo that differs from the perfect match by only a single base in the center position. Probes have been chosen from unique regions in the gene, weighted toward the 3’ end. Each array also contains probe pairs corresponding to multiple reference genes, allowing standards to be added to the sample prior to hybridization for normalization and quantitation of data from different experiments. Arrays are available for human, mouse, rat, E. coli, yeast, and Arabidopsis species, with regular updates as sequence information increases.
Total RNA is provided by the investigator; this is used as a template to generate ds cDNA and, ultimately, biotinylated cRNA. cRNA is hybridized to a chip under appropriate conditions; the chip is washed and incubated with phycoerythin coupled to streptavidin. The arrays are scanned using a scanning confocal microscope; this generates a visible hybridization pattern and quantitative data. The data are analyzed by calculation of the specific hybridization (perfect match-mismatch pairs) and averaging of the signal intensity for 18 of the 20 probe pairs for each gene. (The highest and lowest values are not included to avoid spurious signals.)
This allows quantitative and reproducible detection over a wide range of mRNA expression levels for all of the genes represented on the chip and permits detection of expression of a gene present at 1:100,000 or less (5-10 copies of the transcript per cell). A change of 2-fold or greater between samples is considered significant.
Equipment:
GeneChip® Scanner 3000
Fluidics Station 400
Hybridization Oven 640
Types of array analysis supported:
3' Expression arrays
Exon arrays
Promoter arrays
Services of the Joslin Genomics Core include:
1. Preparation of cRNA for hybridization - from total RNA provided by investigator - with spike-in controls
2. Preparation of hybridization cocktail, including internal controls
3. Hybridization of cRNA samples to test arrays and/or experimental arrays
4. Assistance with sample preparation (RNA isolation), experimental design, and choice of chips
5. Preliminary analysis of data with GCOS 1.4
6. Secure data storage, accessible only by investigator/fellow, on our GCOS database
7. Access to data analysis and bioinformatics tools available in Genomics Core and tutorials upon request
8. Access to other bioinformatics tools and collaboration with bioinformaticians, as part of the BIDMC/JDC/Children's shared NIDDK Biotechnology Center.
Please contact Chris Botka, Bioinformatics Core Director, for assistance with analysis options and tools.
9. Assistance with steps towards validation of genes of interest through RT-PCR. Please see the PCR core website.
How do I get started?
Identify appropriate control and experimental samples. Discuss your project with core personnel, including choice of chip to be used for hybridization. Isolate high-quality total RNA, quantitate it, and verify quality by gel electrophoresis. Submit online order, bring samples and chips (and gel electrophoresis photograph, if applicable) to Genomics Core.
Recommended:
The Genomics Core will now be providing spike-in controls for GeneChip® Expression Profiling. Affymetrix offers a Eukaryotic Poly-A RNA Control Kit which provides positive controls that are spiked into RNA samples prior to cRNA synthesis. While the core lab has always used hybridization controls, these controls permit monitoring of the entire target labeling process. Now you can evaluate assay sensitivity, consistency, and dynamic range. Core users can come by room 607 or e-mail to ask for aliquots of the spike-in controls. Please provide amount of starting total RNA you have for each sample.
How much will this cost?
Please email core personnel for current cost of Affymetrix chips (with academic discount). At present, experimental chips will be purchased by the investigator. The new GeneChip scanner is compatible with the new 2.0 arrays. We highly recommend investigators to use the 2.0 arrays since they combine A and B arrays into one resulting in a substantial savings.
Data analysis and Bioinformatics Tools:
There are several tools available in the Genomics Core. Several tools are freely available on the internet. Please feel free to inquire about these tools or any other tools you would be interested in. We would be more than happy to provide an overview tutorial of the analysis tools available.
Please contact Chris Botka, Bioinformatics Core Director, for assistance with analysis options and tools.
Also for more resources, please visit: DGAP Resources
Tools available include:
GeneSpring 7.2 GeneSpring is available in the Genomics Core. Please contact Core personnel to setup a time to use the program.
DChip
chipSHARP
R BioConductor
GenMAPP v2
MAPPFinder v2
Onto-Tools
SAM
Broad Institute Software Tools
Broad GSEA
Ingenuity Pathway Analysis (By license only)
Permuted Correlation Analysis (in Core only)
Selected Affymetrix technology references:
Wodicka L et al. Genome-wide expression monitoring in S. cerevisiae. Nature Biotechnology 1997; 15: 1359-1367.
DeSaizieu A et al. Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nature Biotechnology 1998; 16: 45-48.
Alon U et al. Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays. PNAS 1999; 976: 6745-6750.
Fambrough D et al. Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 1999; 97: 727-741.
Cargill M, et al. Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nature Genetics 1999; 22: 231-238
Gerhold D et al. DNA chips: promising toys have become powerful tools. TIBS 1999; 24: 168-173.
Nadler ST et al. The expression of adipogenic genes is decreased in obesity and diabetes mellitus. PNAS 2000; 97: 11371-11376.
Butte AJ et al. Discovering functional relationships between RNA expression and chemotherapeutic susceptibility using relevance networks. PNAS 2000; 97: 12182-12186.
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