Affymetrix Genechip Core Facility
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 high by some, 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|
* 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.
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(2 cycle) hr]. Please contact the lab manager for details.
GeneChip Hybridizations and Scanning (Partial service)
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 – Yoon Gi (Justin) Choi, or 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.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 (partial or full) 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-5:00pm. 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) and RMA quality control files (Box plots, Histograms, RMA estimates).
Note: The FGL may offer custom services for target preparation.
Statistical and Computing Resources
The FGL offers statistical consulting services by the lab manager, Justin Choi. Researchers involved at any stage of a microarray study are encouraged to take advantage of one hour of free consultation per project. Additional services will be billed at $75.00 per hour on a recharge basis. We can provide any level of advice, training, or execution of the following technical services for either spotted array or Affymetrix platforms:
Microarray study design
Simple and complex
Microarray data extraction and quality analysis
- Spot – spotted array gene expression quantification program using sophisticated image analysis and spot detection algorithms
- GenePix – spotted array gene expression quantification and quality assessment software
- RMA – gene expression quantification software based on models simultaneously fitting multiple Affymertrix GeneChips
- Affy MAS5 – Affymetrix proprietary gene expression quantification and quality assessment software
Significance testing, multiple testing adjustment, false discovery rates, time course analysis
- Hierarchical Clustering – clustering method to detect order among genes (or array samples) and create trees based on pair-wise similarity between genes
- K-means – assignment of genes (or array samples) into k clusters of nearest neighbors
- HOPACH – hybrid clustering method, hierarchical ordered partitioning and collapsing hybrid (HOPACH), which is a hierarchical tree of clusters
- SOM – self-organizing maps, partitions genes into groups with similar expression patterns
Pathways and Gene Ontology
GenMAPP – software to visualize gene expression data on metabolic and Gene Ontology maps
- Bioconductor – an open source suite of microarray analysis programs for both spotted array and Affymetrix GeneChip experiments
Data Storage, Database
GeneTraffic – web-based database to house and analyze microarray data of both spotted and Affymetrix types
Statistical interpretation and writing
Computer system resources offered by the Functional Genomics Lab
The FGL supports a Windows 2003 Server and Domain. Users are given a personal password-protected account on the FGL domain. Data and desktop are securely stored on the server and are accessible by any user logging onto the domain from any of our four public computers in the FGL. The server is firewalled and protected by nightly backup. One computer is reserved for scanner and GenePix users. The other three have all software necessary to do microarray analyses and standard Microsoft Office applications.
The FGL supports Gene Traffic, a web-based microarray database and analysis server. From any computer with Internet Explorer and an internet connection, a user can upload image and/or quantification files to the Gene Traffic server into a permanent project dataset. Data projects are linked to visualization, statistical, clustering tools, and extensive gene annotation, providing biologists easy discovery of differentially expressed genes and their biological connections. For a nominal fee, registered users are entitled to unlimited access to the Gene Traffic server, training, and support. Please contact the lab manager for current recharge rates.
- 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.