Illumina BeadChip

Location: Thomas Bldg DE-740
Contact phone: (206) 667-2714
Contact fax: (206) 667-2825
Contact e-mail: genomics@fhcrc.org

Lab Introduction and Consultation

Illumina's Genome-Wide Expression BeadChip product line includes a selection of array types for human, mouse, and rat expression profiling. All expression BeadChips are designed for single-channel transcriptome analysis and are intended for single-use. In addition, Illumina offers focused arrays for expression analysis, as well as a product for microRNA expression profiling. For an overview of the Illumina RNA Analysis platforms and product lines, please visit their website.

The DNA Array laboratory has an Illumina BeadStation System, which includes two BeadArray readers and supporting automation (chip autoloader and a Tecan liquid-handling robot).

The following are instructions on how Fred Hutchinson and other Cancer Consortium members can access Illumina RNA Analysis services at FHCRC, with an emphasis on whole-genome expression profiling. Those interested in focused expression analysis or microRNA profiling should contact the lab for additional information. In addition, those new to this technology or new to using the Genomics Resource are strongly encourage to meet with lab personnel prior to planning a project. For non-consortium members interested in services please contact the Genomics Resource at (206) 667-2714.

  • For questions regarding experimental design issues, please contact Jeff Delrow at jdelrow@fhcrc.org or (206) 667-2763.
     
  • For questions regarding service fees, scheduling, and protocols please contact Alyssa Dawson at adawson@fhcrc.org or (206) 667-5614.

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Reagent and Consumable Purchases

Each investigator is required to purchase Illumina Expression arrays and reagents directly through Illumina. The Genomics Resource does not stock Illumina products. Please contact Illumina for pricing.

Illumina Sales Contact:

Robin L. Ball
Regional Account Manager
Phone: 206-660-7749
Email: rball@illumina.com

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Service option 1: Total RNA

Our sample requirements for expression profiling using Illumina expression arrays and for QC analysis are >400 ng of total RNA at a concentration of at least 100ng/μl. While the lab will perform QC analysis of all sample submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Failure to perform standard QC analysis of your RNA samples will result in sample processing delays.

All samples, Illumina BeadChips, Illumina BeadChip processing reagents, and a fully completed sample submission form should be delivered to the Genomics Resource, located in the Thomas Building, DE-740.

Illumina BeadChip request forms
If you are using an FHCRC budget number, please fill out the Internal Request Form.

If billing is to be submitted to the University of Washington or other external institution, fill out External Request Form.

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Service option 1: Sample QC and Labeling

Quality control of total RNA includes the following:

QC test for RNA degradation. The resource has two Agilent 2100 Bioanalyzers that are used to assess the initial RNA quality. To learn more about how to interpret Bioanalyzer results, please visit the Agilent web site.

QC test for protein and organic contaminates. The resource has a NanoDrop DN-1000 spectrophotometer that is used to assess sample purity. Employing a UV scan from 210 - 310 nm, the following criteria must be met:

  • 1.8 ≤ A260 / A280 ≤ 2.1
  • A260/ A230 ≥ 1.7
  • A310 ~ 0

To ensure that the above criteria are based on sufficient absorption measurements, we also require an A260 > 0.3 when performing this analysis.

In instances where one or more samples do not pass our initial QC tests, we will not proceed with the project and immediately contact the submitter with our findings.

The Genomics Resource staff generate biotin-labeled cRNA targets for hybridization with strict adherence to the sample preparation protocols outlined in the Illumina TotalPrep RNA Amplification Kit Instruction Manual. Samples will be closely monitored throughout this process using the Agilent 2100 Bioanalyzer and the NanoDrop ND-1000 spectrophotometer.

In instances where one or more samples do not pass our QC tests, we will not proceed with the project and immediately contact the submitter with our findings.

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Service option 2: Labeled cRNA Cocktail

Biotin-labeled cRNA sample requirements for expression profiling using Illumina Expression BeadChips are:

  • 6-sample BeadChip: 1.5ug of biotin-labeled cRNA in a total volume of 10ul
  • 8 or 12-sample BeadChip: 750ng of biotin-labeled cRNA in a total volume of 5ul

For labeling protocols please refer to the llumina TotalPrep RNA Amplification Kit Instruction Manual. The Genomics Resource lab will not perform QC analysis of biotin-labeled cRNA samples submitted for microarray analysis, so it is advisable that the investigator perform assessment of sample quality before sending samples to the lab.

All samples, Illumina BeadChips, Illumina BeadChip processing reagents, and a fully completed sample submission form should be delivered to the Genomics Resource, located in the Thomas Building, DE-740.

Illumina BeadChip request forms:
If you are using a FHCRC project id for billing, please fill out the Internal Request Form.

If billing is to be submitted to the University of Washington or other external institution, fill out the External Request Form.

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Array Processing

Strictly adhering to protocols outlined in the Whole-Genome Gene Expression with IntelliHyb Seal Experienced User Card and the Illumina Whole-Genome Gene Expression with IntelliHyb Seal System Manual,* the Genomics Resource Lab will hybridize the biotin-labeled target overnight to the Illumina Expression BeadChip. Following hybridization, the BeadChip undergoes a washing and streptavidin-Cy3 conjugate staining protocol. Once the BeadChip has been processed it is scanned by the Genomics Resource Lab with an Illumina BeadArray Reader.

*Please note that the System Manual is out of date and Illumina has not provided an up-to -date manual. So, where there are differences in the protocols outlined in the Experienced User Card and the System Manual, the Experience User Card is followed.

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Results/Downstream Analysis

Data generated within the resource are transferred through automated pipelines to systems supported by the Hutchinson Center's IT department. Data is transferred to the user's 'fred' account, in the researcher's dnaarray folder.

If you do not have a ‘fred’ account, please submit the computing account application form.

Each image is stored as an image data file (.idat extension) in a folder labeled with the BeadChip barcode number. Each BeadChip is accompanied with a disk which contains the Decode Data (.dmap extension) also in a folder labeled with the BeadChip barcode number, the Array Content Directory (.xml or .bgx extension), and the Bead Set Manifest (.csv extension).

While the lab will perform QC analysis of all microarrays processed, it is advisable that the investigator perform an assessment of microarray quality before proceeding to further analysis.

Researchers may transfer their BeadChip files from the FRED server into the Illumina BeadStudio Application where gene expression data can be extracted from the images collected from the Illumina BeadArray Reader. Resulting files can be exported to most standard databases and gene expression analysis programs. Please contact Ryan Basom to gain access to the BeadStudio software.

Support is available through the Genomics Resource to those needing assistance with analyzing and interpreting their data.  Additional data analysis support is also available through the Computational Biology Shared Resource.   For more information, please contact:

Jeff Delrow, Ph.D.
Director and Staff Scientist, Genomics Resource
(206) 667-2763
jdelrow@fhcrc.org

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Doing Business with Genomics

Thomas Bldg DE-740
(206) 667-2714
genomics@fhcrc.org

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