Agilent RNA Analysis

   

Agilent Technologies expression array product line includes a large selection of cataloged, 60mer isothermal oligo arrays covering many different organisms. Custom array designs are also possible. All expression arrays are designed for dual mode (also referred to as two-channel or two-color) transcriptome analysis and are intended for single-use. For an overview of the Agilent microarray platform and product line, please visit their website.

The DNA Array Lab performs Agilent array analysis using Robbins Scientific hybridization ovens, each fitted with an Agilent rotator rack. In addition the lab has an Agilent Technologies array scanner with a 48-position carousel.

The following are instructions on how Cancer Consortium members can access Agilent Technologies microarray services at FHCRC. 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 Genomic Resource at (206) 667-2714.

Contacts:

• 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 Cassie Sather at csather@fhcrc.org or (206) 667-2757.

   

Each investigator is required to purchase arrays directly through Agilent. The Genomics Resource does not stock Agilent arrays. A list of current array pricing can be obtained from the sales contact listed below.

Agilent Sales Contact:

Angelyn Regala
Product Specialist Integrated Biology Solutions
1-408-553-3733
angelyn_regala@agilent.com

   

Total RNA sample requirements for two-color expression profiling using Agilent arrays are 4 ug of total RNA at a concentration of at least 200ng/ul. While the lab will perform QC analysis of all samples 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, Agilent arrays, and a fully completed sample submission form should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740.

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.

   

Quality control of total RNA includes:
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 ND-1000 spectrophotometer that is used to assess sample purity. Employing a UV scan from 210nm - 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 will generate Cy3 and Cy5 cRNA targets for hybridization with strict adherence to the sample preparation protocols outlined in the Ambion Message Amplification Manual and the Agilent Two-Color Microarray-Based Gene Expression Analysis Manual.

The specific procedures are detailed in the Agilent Labeling Protocol.

Samples will be closely monitored throughout this process using the Agilent 2100 Bioanalyzer and the NanoDrop ND-1000 spectrophotometer.

   

Labeled cRNA sample requirements for expression profiling using Agilent arrays vary depending on the array type. Both the submission volume and submission amount will vary. In all cases, however, only unfragmented samples should be submitted. Samples should be submitted in water only. Fragmentation will be performed by the Genomics Resource staff. Additionally, Genomics Resource staff will provide and add the blocking agent and the hybridization buffer. For water volume and sample amount requirements, please see Page 27 of the Agilent Two-Color Microarray-Based Gene Expression Analysis Manual for your particular array format requirements.

Additionally, we highly recommend that researchers monitor their samples throughout the labeling process. It is generally recommended that samples meet a minimum frequency of dye-incorporation of >15. More information on this topic can be found in the NanoDrop ND-1000 Microgenomics Application Note.

All samples, Agilent arrays, and a fully completed sample submission form should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740.

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.

   

Strictly adhering to procedures outlined in the Agilent Two-Color Microarray-Based Gene Expression Analysis Manual, the Genomics Resource Lab will hybridize the labeled target overnight to the Agilent Expression array. Following hybridization, the array undergoes post-hybridization washes outlined in the Agilent Two-Color Microarray-Based Gene Expression Analysis Manual.

   

Once the Agilent array has been processed it is scanned by the Genomics Resource Lab to generate a TIFF image file. The lab is equipped with an Agilent G2565BA Microarray Scanner System.

Using the associated Agilent Feature Extraction Software images are automatically processed and intensity files are generated. For more information on the files generated, please refer to the Agilent Feature Extraction Output Description File .

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

   

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

If you do not currently have an account on the Research Computing server please fill out the User Account Application prior to the completion of your microarray experiment.

   

For more information on downstream data processing and storage please see our informatics web page.

For further processing and analysis of your microarray data, there are many third party software applications that can be used. Some of the software applications available to you can be found on our software list or the Genomics IT web page. (License agreements dictate that some of the software is available only from FHCRC based computers. If you are not at FHCRC and need access to the software, please contact Genomics and we will arrange access for you.)

Additional support is available to those requiring assistance with analyzing and interpreting their data. Please contact one of the following for guidance:

Jeff Delrow, Ph.D.
Director and Staff Scientist, Genomics Resource
voice : 206.667.2763
email : jdelrow@fhcrc.org

Martin Morgan, Ph.D.
Director and Staff Scientist, Computational Biology Shared Resource
voice: 208.667-2793
email: mtmorgan@fhcrc.org