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|  Oregon Health and Science University |
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| Oregon Health and Science University |
The OHSU Gene Profiling Shared Resource (formerly the Gene Microarray Shared Resource) functions as a full-service genomics facility serving research scientists and clinicians with services for RNA expression profiling and DNA variation analysis on two microarray platforms, Affymetrix and Illumina and on Applied Biosystems QuantStudio real-time PCR system. We are also now able to provide expression profiling and genome analysis services using Ion Torrent PGM sequencer. Support for RNA isolation and RNA/DNA quality assessment also available.
Designed for use with Affymetrix GeneChip expression probe arrays and Genotyping assays. The system includes the two-color GeneChip Scanner 3000 with AutoLoader, three Fluidics Station 450s, Hybridization Oven 645, a powerful computer workstation with Affymetrix GeneChip Operating System (GCOS) and GeneChip Command Console Software (AGCC), and scanner workstation with a 2D handheld barcode reader.
"The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells on a single platform. Results are delivered within 30-40 minutes in automated, high quality digital data.
Agilent's 2100 Bioanalyzer uses a lab-on-a-chip approach to perform fluorescence-based capillary electrophoresis to assess starting sample integrity. A concentration measurement is also generated, which is compared against optical density (OD) measurements obtained from the NanoDrop-1000/8000 spectrophotometer. The Bioanalyzer uses two chip types: the Nano LabChip (and the Pico LabChip."
"The LabChip GX family of instruments is the most advanced nucleic acid and protein separations system available today. Like its predecessor the LabChip 90, the GX utilizes PerkinElmer's innovative microfluidics technology to perform reproducible, high-resolution, eletrophoretic separations. Whether analyzing RNA integrity for better gene expression data or assessing protein quality in biological fermentation, the LabChip GX instrument accelerates your research and helps you generate more meaningful data, faster."
"The epMotion 5075 LH is the ideal solution for advanced liquid handling demands. It offers the same outstanding accuracy and precision as epMotion 5070, making it an excellent tool for demanding, small-volume applications such as real-time PCR set-up or magnetic bead purification, as well as any routine pipetting task.
The 12 positions and the automatic tool exchange expand the application range to handle complex patterns and higher sample numbers. With heating and cooling and the gripper option, the epMotion 5075 LH is one of the most flexible and future-proof automated pipetting systems available."
"The Illumina BeadReader scanner is designed to read both slide-based arrays and the multi-well Sentrix Array Matrices. It has <1 micron resolution and can simultaneously scan at two wavelengths. Excitation wavelengths are 532 nm and 635 nm. The emission spectrum is 550-600 nm and 650-700 nm. Files are output in 16-bit TIFF images. The limits of detection are 2 fluor/sq. micron. The IMC scanner has been modified by Illumina to allow scanning of high density arrays."
There are multiple machines.
Multi-detection microplate reader with dual-monochromators and dual-mode cuvette ports. The system can detect absorbance (UV-Vis Abs) and fluorescence intensity (FI) and can read 6- to 384-well microplates.
"For fully automated sample prep using spin-column kits
- Automation of trusted QIAGEN spin-column kits
- Elimination of manual processing steps
- Purification of DNA, RNA, or proteins
- More free time with affordable automated processing
- Standardized results and increased productivity"
Lab has two of these instruments.
"This document provides a brief description of methods used in the AMC laboratory for sample labeling and array hybridization and processing during the period of June 2004 through September 2007 for GeneChip arrays with feature sizes greater than or equal to 11-micron (e.g, HG-U133 Plus 2.0,MOE 430 2.0)."
"Need to decode an AMC ID? This links to information about how the AMC names hybridizations and why the schema is used."
"This document provides a brief description of methods used in the AMC laboratory for sample labeling and array hybridization and processing during the period of Jan 2002 through June 2004 for GeneChip arrays with feature sizes greater than or equal to 18 micron (e.g, HG-U133A, MOE430A, MG-U74A). Note: for sample inputs of less than 4μg or assays with GeneChip 2.0 all-on-one arrays, the protocols described in this document are not accurate."
"Protocols vary depending upon the array being run, so it is wise to check with the IMC to see how the following applies to a specific product.
DNA is prepared by standard high quality protocols. 200 to 750 ng is needed for each sample, depending upon the size of the array (1). The DNA is denatured and then neutralized for amplification. The denatured DNA is isothermally amplified several thousandfold using a proprietary Illumina protocol. The amplified product is fragmented by a controlled enzymatic process that does not require gel electrophoresis. Overfragmentation is avoided by using end-point fragmentation. Fragmented DNA is recoved by isopropanol precipitation. The fragmented DNA is then hybridized to the 50-mer probes of the BeadChip.
Unhybridized and non-specific DNA is washed from the array. Single base extension of the oligos on the BeadChip incorporates modified nucleotides at the site of the single nucleotide polymorphism. The labeled nucleotides include biotin-labeled ddCTP and ddGTP and 2,4-dinitrophenol (DNP) -labeled ddATP and ddUTP. Following additional washing to remove unincorporated nucleotide, the arrays are stained with Alexa555 and Alexa 647 using a dual color, orthogonal, multi-layer immunohistochemical sandwich assay (2). Stained arrays are scanned using the Illumina BeadReader microarray scanner. The data is consolidated using Illumina's GenomeStudio software."
Total RNA is submitted by the investigator to the Illumina Microarray Core (IMC). The concentration of the RNA is verified by the IMC using a NanoDrop spectrophotometer. This step also looks for the appropriate 260 / 280 ratio for RNA and looks for organic and salt carryover in the sample using the 260 / 230 ratio. An aliquot of each sample is run on an Bioanalyzer (Agilent) to observe 18S and 28S ribosomal RNA peaks and to look for degradation of the RNA.
Total RNA that has passed the QC tests will be amplified using the TotalPrep RNA Amplification Kit (Ambion), which can amplify RNA across a range from 50 to 500 ng. The resultant amplified RNA (aRNA) incorporates a biotinylated UTP nucleotide. An aliquot of aRNA is hybridized to an Illumina BeadChip using proprietary buffers, first at 65⁰C for thirty minutes, then overnight at 58⁰C.
Following hybridization, the arrays are washed and then stained with Cy3-streptavidin, washed again, and then scanned on an Illumina BeadStation bead array scanner. Data from the scanner is consolated using the GenomeStudio software. Built in quality control measures are monitored in GenomeStudio to look for obvious problems in the hybridization process. Final output is customized to allow use of a variety of statistical packages, including BioConductor and GeneSifter.
"Data Preparation includes the following steps:
1. Visual inspection of all raw image scans
2. Visualization of overall distribution and/or sample correlation by data plots of unscaled, non-normalized data
3. Assessment of standard array quality metrics, which are output in Excel format
4. Preparation of a Project Report, which includes assay processing details as well as a core project assessment, description of all output files, and a listing of core and external post-project support options
5. All array data files are provided; scaled/normalized data files are also provided for expression projects" "Data Security
Strict policies surround data security in the Affymetrix Microarray Core. Project correspondences and project data are released only to investigators that are listed on the Project Application and Sample Submission Form. A principal investigator may add/delete an investigator from the project forms by e-mailing Michelle Garred, Affymetrix Microarray Core/GMSR User Support (garredm@ohsu.edu)." "Data Management
The Affymetrix Microarray Core practices data redundancy for an increased level of data security. Within the core, raw array data files are maintained on a local drive and raw, processed, and quality assessment (QA) data is maintained in the disc archives. Also, all data that is delivered to the investigator's lab is transferred to a server that is maintained by OHSU's computing group, AGCC. These storage steps, along with the copy of data that is distributed directly to the investigator's lab offers a significant degree of data stability.
Note: The Affymetrix Microarray Core is not responsible for lost data post-project completion. The investigator's lab is encouraged to practice redundant back-up measures to protect against lost/corrupted data discs, drive failure, etc."
Basic statistical analysis and data QC of microarray and PCR data.
Further data analysis performed in collaboration with the Knight Cancer Institute's Biostatistics Shared Resource and Bioinformatics Shared Resource.
"Whole Genome Genotyping Arrays: Human660W-Quad - More than 658,000 markers with an median spacing of 2.5 kb. Each slide assays four samples with as little as 200 ng of starting genomic DNA. Illumina HD arrays can achieve an average call rate of greater than 99%. The Human660W-Quad content is based on HapMap release 23. It offers high value coverage across many populations, captures the majority of known variation, and targets common CNV regions. It contains about 100,000 markers that specifically target regions of common copy number varation that were recurrently identified in a high-density CNV discovery project across mutiple population."
"Illumina offers a variety of arrays for genotyping for focused human studies, for non-human samples and for methylation analysis. Please contact the core director or visit the Illumina website for more information. Briefly, among the focused arrays available are:
African American admixture panel
Cancer SNP panel
HumanCVD panel
MHC panel
Bovine, canine, equine, ovine, and procine arrays
In addition, custom arrays can be designed.
Methylation arrays includes the infinium methylation array and the cancer methylation panel, plus the option of creating a custom methylation panel."
"mRNA and microRNA expression profiling, including options for whole transcript analysis and alternative transcript/exon analysis."
The GMSR welcomes project applications from OHSU and non-OHSU investigators (academic and commercial).
"Assistance with determining RNA/DNA concentration, sample clean-up and sample concentration is available through the core lab.
Due to the experimental nature of microarray projects, samples may fail or need to be reprocessed. When this happens, the core will inform you if additional charges are necessary.
All prices quoted on this website reflect OHSU investigator/affiliate pricing. The Affymetrix Microarray Core accepts projects from non-OHSU investigators from both non-profit and commercial organizations. A 15% service surcharge will be applied for non-affiliated academic/non-profit customers and for all projects not paid for through an OHSU administered grant or account. For services and pricing available to commercial organizations, contact the Core Director."
"The concentration of samples submitted to the Affymetrix Microarray Core is measured on a dedicated NanoDrop-1000 or NanoDrop-8000 spectrophotometer that is performance checked using DNA standards prior to collecting sample measurements. Using concentration data provided by the investigator's laboratory, the most accurate method is determined for quantification of the RNA/DNA sample. Currently we routinely use both standard absorbance and fluorometric techniques.
The integrity of samples submitted to the Affymetrix Microarray Core is assessed using the Agilent 2100 Bioanalyzer, which uses a chip-based approach to perform fluorescence-based capillary electrophoresis. A concentration measurement is also generated, which is compared against optical density (OD) measurements obtained from the NanoDrop spectrophotometer.
The goal of a sample quality assessment is to determine the quantity and quality of RNA/DNA sample material prior to performing the microarray assay."
Can also be setup to make use of Nanodrop 3300 fluorospectrometer from the DNA services core.
"Microarray DNA Variation Analysis:
DNA Labeling Method
- Affymetrix 250K
- Affymetrix SNP 6.0 (Standard)" "RNA profiling:
* 3'IVT Labeling method
- Ambion MessageAMP Premier (Standard)
- Affymetrix One Cycle cDNA synthesis
- NuGEN Ovation Labeling
* Whole Transcript Labeling for Gene and Exon arrays
- Ambion Whole Transcript Sense target labeling (Standard)
* miRNA labeling for Affymetrix miRNA array
- Genisphere High Sensitivity FlashTag Biotin labeling" "Notes:
- If the concentration of samples is unknown, please contact the Gene Microarray Shared Resource lab before submitting samples.
- When submitting samples, RNA should be in an RNAse, DNAse-free 1.5ml microcentrifuge tube and in aqueous solution, preferably nuclease-free water.
- When submitting samples with small RNA inputs (5-100ng total), please contact the Gene Microarray Shared Resource lab before submitting samples for any special considerations.
- For any sample preparation and sample submission questions, contact the Gene Microarray Shared Resource lab.Reduced pricing is available for batch processing of 24 or more samples.
- To submit polyA+ RNA for expression profiling, please contact the Gene Microarray Shared Resource lab for submission requirements."
"Affymetrix® GeneChip® Command Console® Software (AGCC) is the latest generation of instrument control software for GeneChip systems. Command Console Software provides an intuitive set of tools for instrument control and data management used in the processing of GeneChip Arrays. The software summarizes probe cell intensity data (CEL file generation) and enables sample and array registration, data management, instrument control as well as automatic and manual image gridding. Besides these core features, the flexible platform provided by Command Console Software enables customized, automated, and integrated workflows with a variety of laboratory information management systems. Probe-level summarization (CHP) files are generated by other Affymetrix software applications."
Macros and scripts are used for preliminary processing for any of the 3 prime arrays.
"GTC Software integrates single nucleotide polymorphism (SNP), copy number polymorphism (CNP) genotyping, rare copy number variation (CNV) identification, and cytogenetic analyses into one application. It generates genotyping calls, copy number calls for CNV regions and individual probe sets, loss of heterozygosity (LOH) data, cluster graphs, and quality control metrics."
Macros and scripts are used for preliminary processing for any of the 3 prime arrays.
"Affymetrix® Microarray Suite (MAS) software provides instrument control for the GeneArray® Scanner and Fluidic Station, array image acquisition and analysis, and communication with Affymetrix® LIMS software for data storage and management. All functions are clearly displayed in the user interface using multiple toolbar displays, enabling you to rapidly locate and analyze GeneChip® array data."
"Bioconductor provides tools for the analysis and comprehension of high-throughput genomic data. Bioconductor uses the R statistical programming language, and is open source and open development. It has two releases each year, more than 460 packages, and an active user community."
"GeneSifter offers a robust statistical framework with 15 advanced options, including 2-way ANOVA, PCA, PAM, hierarchical clustering, CLARA, FDR, FWER, RMA, Max T and more to analyze microarray data."
"Visualize and analyze data generated by all of Illumina's platforms with GenomeStudio Data Analysis Software. This powerful informatics solution supports the primary analysis of sequence-based data produced by the Genome AnalyzerIIx and microarray-based data generated by the iScan System and BeadXpress Reader. Performance optimized tools and a user-friendly graphical interface allow you to quickly and easily convert data into meaningful results for your genome analysis and research."
"MetaCore™ is an integrated knowledge database and software suite for pathway analysis of experimental data and gene lists. The scope of data types includes microarray and sequence-based gene expression, SNPs and CGH arrays, proteomics, metabolomics, Co-IP pull-out and other custom interactions. MetaCore™ is based on a proprietary manually curated database of human protein-protein, protein-DNA and protein compound interactions, metabolic and signaling pathways for human, mouse and rat, supported by proprietary ontologies and controlled vocabulary. The analytical package includes easy to use, intuitive tools for search, data visualization, mapping and exchange, biological networks and interactome."
"Partek® Genomics SuiteTM is a comprehensive suite of advanced statistics and interactive data visualization specifically designed to reliably extract biological signals from noisy data. The software is unique in supporting all microarray and next generation sequencing technologies including gene expression and digital gene expression (DGE), exon/alternative splicing and RNA-Seq, copy number and association, ChIP-chip, ChIP-seq, and microRNA in a single software package, allowing for analysis of multiple applications in one complete solution."