Affymetrix GeneChip Services
With the advent of NGS and high throughput arrays, GeneChips are seeing decreased usage. However, we maintain this service for our legacy users.
We utilize 3′ IVT PLUS Reagent Kit for the production of biotinylated cRNA hybridization probes.
The 3′ IVT Express kit requires 50-500ng of total RNA. We ask that you submit 1µg of total RNA at a concentration of about 200ng/ul or higher.
We utilize the Whole Transcript dsDNA Target Assay, which requires 7ug total RNA/array. We ask for 10ug total RNA at 1ug/ul.
We utilize the Agilent Whole Transcript Expression Assay for sense strand cDNA probes. This kit requires 50-500ng of total RNA. We ask that you submit 1µg of total RNA at a concentration of about 200ng/ul.
For an overview of the protocols and hybridization methods employed, see the Affymetrix Technical Manuals. For the best results, it is suggested that samples to be compared are produced using the same RNA type (mRNA or total RNA), the same labeling kit, and at approximately the same amount of starting material.
For RNA extraction, it is best to utilize established protocols for particular tissues or cells. For new tissue, Affymetrix lists suggests:
Arabidopsis total RNA: TRIzol reagent (Invitrogen)
mammalian tissue total RNA: TRIzol
mammalian cells: RNeasy Mini kit (Qiagen)
Ethanol precipitation is required following TRIzol and phenol extraction methods. These kits provide consistently good yields of labeled cRNA.
Submission of Samples
Included in the rate for probe labeling, all RNA samples are examined for quality using the Agilent 2100 Bioanalyzer as recommended by Affymetrix. The device uses microfluidics and requires only 50 – 500ng/sample. A small aliquot of all RNA samples submitted will be checked in this manner. There is a modest fee associated with the Bioanalyzer in order to purchase microfluidic chips for samples that must be repeated (for example, degraded samples), so we advise customers to run a 1% agarose gel to check the quality of their RNA samples prior to submission. Please fill in the online sample submission form completely when requesting genechip services. The form can be found at the IIGB Billing and Reservation Site. If you have not used the site, you are required to register online to access services and forms.
Affymetrix offers a wide selection of genechips. For a list of available chips, please visit the Affymetrix website. Chips should be ordered and paid for by users. However, they can be sent directly to the Genomics Core facility at the address indicated below. They should be kept at 4 degrees centigrade, but not frozen.
Data Retrieval and Analysis
A CD containing all x files will be provided. Data can also be uploaded to our Dropbox account and an email containing a link will be sent. You are not required to have a Dropbox account to download data from the link we provide. These files are large and require a lot of data storage space, so they will be deleted after one week. Please call (951) 827-7928 or email firstname.lastname@example.org to request a Dropbox link. Other data files (.cel and .txt) will be provided. The .txt files can be opened using Excel.
The Genomics Core provides all files necessary for analysis of Affymetrix Genechip results. The files can be analyzed by users with the Expression Console Software available for download free by Affymetrix. The .cel files can be used in other analysis software such as Array Assist from Stratagene.
For more detailed analysis of results, users should contact the Bioinformatics Core of IIGB which offers detailed workshops in analysis as well as services for standard and custom applications.
If there is any question concerning the quality of total RNA, a test chip can be purchased to test the quality of the cRNA probes prior to using the more expensive specific chips.
For useful information regarding analysis of Affymetrix Chip results, the GeneChip® Expression Analysis Data Analysis Fundamentals Manual provides useful guidelines.
All non-UCR customers should send their RNA on dry ice to the following address:
ATTN: Holly Clark
University of California
900 University Avenue
Dept. of Botany & Plant Sciences
2150 Bachelor Hall
Riverside, CA 92521
Genechips should be sent to this address on blue ice (not frozen).