UC BERKELEY FUNCTIONAL GENOMICS LABORATORY
AFFYMETRIX GENECHIP CORE FACILITY
Overview
Affymetrix GeneChip microarrays provide a sensitive and reliable technology platform for measuring gene expression levels from tens of thousands of genes simultaneously. Affymetrix GeneChips can also be used for other applications (e.g., SNP analysis). Although the cost per chip is considered by some to be high, this is offset by the quality and reproducibility of the data that can be obtained.
Keys to Successful GeneChip Experiments
The single most important determinant for the outcome of a GeneChip analysis experiment is the quality of the RNA. Therefore, it is essential to check the quality of your RNA sample each and every time you prepare target cRNA.
For comparative gene expression analysis, it is essential that all experimental conditions such as incubation time, temperature, reagents, and sample preparations be identical for all samples.
Target cRNA Preparation
We recommend that you follow the Target Preparation Protocol described in the Affymetrix "Expression Analysis Technical Manual" and use the reagents recommended in the manual, as those reagents have been tested by Affymetrix and are known to work.
Total RNA is preferable to mRNA as starting material to avoid loss of starting material as well as time and cost involved during mRNA purification. The preparation of the target cRNA is both time consuming and expensive and therefore you are well advised to monitor the quality of your RNA sample both before and during the labeled cRNA preparation by using a A260/A280 ratio, agarose gel, or both.
| Sample | A260/A280 ratio | Total RNA |
| Agarose gel | + | + |
| cDNA | - | + |
| cRNA | + | + |
| Fragmented cRNA* | + | + |
* Use the Ambion Century Marker to check the size of the fragmented cRNA (although any 1 kb size ladder should work), which should be between 35 to 200 bases.
- The A260/A280 ratio should fall within the value of 1.9 to 2.0.
- The 28S/18S RNA bands (from the gel) should be present as bands if you are running a gel for total RNA. Bands should be crisp and the intensity of the 28S band should be roughly twice the intensity of the 18S band.
- For small samples (or small amounts of starting materials), it is recommended that you check total RNA yield with a chemical measurement (ie. BioAnalyzer, RiboGreen, etc.).
The minimum amount of fragmented cRNA required for hybridization is:
- 5ug for Micro/Mini array (Test3 array)
- 10ug for Midi array
- 15ug for Standard array
The concentration of fragmented cRNA should be at least 0.5 ug/ul.
When submitting a sample for processing, please include gel pictures of the intact cRNA and the fragmented cRNA ran next to a size ladder. The fragmented cRNA should run between 50bp - 200 bp, if you are using a DNA size ladder
Services Available
-Full service (Total RNA quality check & Labeling with 1 or 2 cycle kit) for GeneChip
We will charge material and labor [1(1 cycle) or 2 (2cycle)hr/chip]. Please contact Jusitn Choi ( ) for this service until we have the approval of the proposed rate (submitted Jan/2008).
-GeneChip Hybridizations and Scanning
The FGL will perform GeneChip hybridization, washing and staining, scanning, and data extraction (your results will be provided to you on a CD). Below are our guidelines for using this service.
- Contact Y. Justin Choi(jygchoi@berkeley.edu, tel. 510-642-1165) for initial consultation and to schedule your run.
- We need your account information before we can process your samples.
Accounting Information for UC Berkeley labs: We need your chartstring (e.g. 1-56639-19900-22233-44-none-none-FN248) or a 10 digit "speedtype" reference to your chartstring.
For those without UC Berkeley accounts: Labs from other UC campuses will need to obtain a P.O. from their departments before submitting the request. Pricing for non-UC Berkeley investigators may include an additional overhead charge (please inquire).
Labs from LBNL are charged the on-campus rate and should have a current purchase order number. Call us if you have any questions.
Others should generate a purchase order and attach it to their order form.
Cost for hybridization, wash, stain and scan: $63/chip (On-campus rate, effective 11/15/2004 - please inquire for off-campus pricing. Prices subject to change.).
- Submitting samples to the facility:
- Fill out the submission form and return it to the facility (via fax or email, or together with your samples).
- Transport your samples (on dry ice) and GeneChips (order directly from Affymetrix) and bring them to 255LSA (Life Sciences Addition) between 8am-4:30pm. Please call ahead to arrange a delivery time. The FGL will not be responsible for samples or GeneChips not delivered directly to FGL personnel (i.e., no unattended drop-offs).
- Submit your labeled fragmented cRNA in RNase-free 1.5 ml eppendorf tubes with names and concentrations written on them.
- Bring OD readings of your total RNA, cRNA and fragmented cRNA (A260, A280, and A260/A280 ratio)
- Bring an agarose gel picture showing total RNA, cRNA and fragmented cRNA.
Each submission should be processed in 3-5 working days, depending on the workload in the FGL. A CD(s) will be returned to you with the requested files (ie. .DAT, .CEL, .CHP, .RPT)
Note: at this time you must prepare the fragmented labeled cRNA yourself; at a later date the FGL may offer custom services for target preparation (RNA extraction and labeling).
Experimental Design and Analysis
The FGL's Principal Statistician, Karen Vranizan, is available for consultation on experimental design and data analysis. The FGL supports analysis of Affymetrix GeneChip data using a number of platforms and software packages, including GeneTraffic (http://microarrays.berkeley.edu/support.php).
GeneChips
- Test arrays: A very good low cost quality control for cRNA preparation and hybridization and can be used to evaluate target quality.
- Standard arrays: Check our Affymetrix price list for currently available arrays (the prices already reflect the UC Pricing)
- Custom arrays from Affymetrix: Contact Affymetrix and coordinate with us.
Please order GeneChips directly from Affymetrix. The FGL does not stock GeneChips for re-purchase.
Contact Information
Contact Justin Choi (jygchoi@berkeley.edu, tel. 510-642-1165) for initial consultation and to schedule your run.
For statistical consulting services, please contact:
Karen Vranizan
, telephone 510-642-7520
For billing information, please contact: Guadalupe Brandon , telephone 510-643-4975
For general administrative support, please contact the individual listed as appropriate.
Last updated 1/20/2005