DBI – Room 246
Contact: Dr. Joan Burnside
The Microarray Core Facility is currently undergoing re-organization. At this time, the Core Facility is not able to accept new projects.
Affymetrix Microarray Experiments
The DBI Microarray Core Facility houses instrumentation to conduct experiments using Affymetrix GeneChip probe arrays. Instrumentation and equipment used to process samples for Affymetrix analysis include: the GeneChip Hybridization Oven 640, the GeneChip Fluidics Station 400, the Agilent GeneArray Scanner, and a computer workstation with GeneChip Operating Software (GCOS). Affymetrix GeneChip oligonucleotide probe arrays are manufactured using photolithographic technology. Human, rat, mouse, Drosophila, Arabidopsis, yeast, and other arrays are available.
Sample preparation for hybridization to the GeneChip arrays includes: isolation of total RNA or purified poly(A)+ mRNA from cells or tissue, synthesis of double-stranded cDNA, biotin-labeling using in vitro transcription to produce labeled cRNA and fragmentation of the cRNA. Next, a hybridization cocktail is prepared containing the fragmented, biotin-labeled sample and probe array controls. The hybridization cocktail is added to the GeneChip probe array and a 16-hour incubation step is carried out overnight in the hybridization oven. Following hybridization, the sample is stained with streptavidin phycoerythrin conjugate in an automated staining and washing protocol using the fluidics station. Finally the probe array is scanned and the data are analyzed using GCOS. For more information regarding the Affymetrix GeneChip platform, please see: http://www.affymetrix.com
Access to Instrumentation
Currently, a self-service option is available for the Affymetrix system. Users who are interested will be trained to use the Affymetrix equipment under supervision. This allows the researcher to perform all of the steps of their Affymetrix experiments from start to finish. Users should contact Dr. Joan Burnside for training.
At the present time, the Core Center does not offer RNA extraction service. Therefore, researchers are responsible for preparing their own RNA samples. Please follow the same link above for the Affymetrix protocols for generating biotin-labeled and fragmented cRNA.
Follow this link to download the Affymetrix GeneChip Expression Analysis Technical Manual, which contains Affymetrix protocols for RNA isolation, synthesis of double-stranded cDNA, biotin-labeling and fragmentation of cRNA, etc.
The GeneChip Test3 Array contains probes targeting the 5’, middle, and 3’ regions of selected housekeeping gene sequences of various organisms. Measurements of signals from these three probe sets provide a way of evaluating the quality of the labeled target prior to analysis on GeneChip expression arrays.
Affymetrix recommends running a test array to evaluate your RNA before proceeding to hybridization on a larger, more expensive GeneChip probe array.