Construction of microarrays is generally dependent on information gained from genome sequencing or EST projects that provide large sets of annotated clones and sequences. A typical array experiment has 4 basic steps. Inserts from cDNA clones are amplified by PCR, spotted and fixed onto specially treated glass slides. RNA from control and treatment samples is isolated and labeled with different fluorescent dyes incorporated during the reverse transcription process. These labeled probes are then mixed and competitively hybridized to the arrayed substrates. A laser scanner is then used to measure the relative intensities of each dye for each individual spot. The ratio of fluorescence levels between the two probes indicates the relative gene expression between the samples

By these processes researchers can evaluate an entire set of genes simultaneously rather than looking at the effects of single genes one at a time. High differential expression of specific genes can then be followed up by conventional means such as northern blot or quantitative RT-PCR. Data from multiple experiments can be combined in order to assign functional information to genes of otherwise unknown function. Genes showing similar expression profiles across differing states are likely to participate in common physiological or metabolic pathways. Cluster analysis programs have been developed which allow detection of co-expressed groups of genes reflecting information on function.

The facility constructs cDNA microarray slides based on fragments of candidate genes provided by the investigator. PCR product for printing is synthesized directly off bacterial culture as template. The investigator can choose to have the PCR reactions, including clean-up, done by our staff or have a graduate student or postdoctoral fellow work in the Functional Genomics lab under the supervision of the Director, Dr. Mark Band. The Functional Genomics Lab provides space, all consumables and equipment, initial training and temporary freezer space. The investigator is charged per plate processed, success or failure.

70 mer oligonucleotides have recently become the template of choice for many microarrays. Oligo synthesis can be ordered from a number of providers. The facility will aliquot appropriate concentrations and resuspend in printing buffer using the Beckman Biomek FX liquid handling robot.

Printing (spotting) of slides, including slide preparation, and setting up the software, will be done by staff of the Functional Genomics lab. A set of 100 slides is printed per run robotically with a GeneMachines OmniGrid 100 microarray printer.

Hybridization reactions will be performed by the investigator. Facility staff will provide protocols, initial training and consultation. A hands-on microarray workshop is offered semi-annually by the center. For details contact Dr. Mark Band (markband@illinois.edu)

Scanning of slides can be performed in the facility after a user account has been created.

Functional Genomics Unit
Mark Band, Ph.D - Director
356 Edward R. Madigan Laboratory, 1201 W. Gregory Drive, Urbana, IL 61801
Phone: (217) 244-3930     FAX: (217) 265-5066      Email: markband@illinois.edu