University of Tennessee Affymetrix Core Facility is a “user fee” facility catering to inhouse and local users.
The facility is located in the Department of Nutrition in the Jessie Harris Building.
The Core is supported by The University of Tennessee’s College of Education, Health
and Human Sciences, The Institute of Agriculture, The School of Veterinary Medicine,
The Department of Biochemistry and Molecular Biology, The Department of
Microbiology, The College of Nursing and The UT Medical College of Knoxville
Division of Surgical Research.
Using Affymetrix Gene Expression GeneChips, investigators are able to examine
differential gene expression in a wide variety of cell types and tissues. There are
currently over a dozen types of GeneChips available including, Yeast, Human, Rats,
Mice. In the upcoming year, over 125 microbial species will be available and plant types
including wheat, corn, and poplar. Studies at other institutions have utilized GeneChip
Expression to study leukemia, lymphomas and solid tumors. Investigators at the
University of Tennessee have used GeneChips to study obesity and lipid metabolism.
The GeneChip probe arrays are manufactured using technologies that combine
photolithography and combinatorial chemistry. Over 400,000 unique oligonucleotide
probes are synthesized on each GeneChip representing anywhere from 12,000 to 23,000
gene/EST. Each probe set contains a perfect match and a single base mismatch to serve
as a control for nonspecific binding.
The Core has the latest Affymetrix GeneChip Instrumentation including a GeneChip 640
Hybridization Oven, a GeneChip 450 Fluidics Station and a GeneChip 3000 Scanner.
The facility also has an Agilent 2100 Bioanalyzer.
The GeneChip 640 Hybridization Oven is capable of hybridizing 64 chips at a time in a
rotisserie fashion for thorough hybridization across the entire array. The GeneChip 450
Fluidics Station contains four wash modules each independently controlled and
monitored with state of the art conductance sensors to accurately check the fluid levels in
the chips.
The GeneChip 7G Scanner uses the Affymetrix patented “flying objective” lens (FOL)
technology which addresses previous scanner’s limitations by increasing scan speed,
decreasing optical distortion and ensuring emission path alignment robustness. The
system uses a high-quality, solid state 532nm Diode-Pumped, Frequency Double Nd:
YAG (Neodynium-doped Yttrium Aluminum Garnet) Green Laser. The resolution of
this laser beam is an excitation spot size of 3.5um. GeneChip features are currently set at
11um. The GeneChip 7G Scanner is able to distinguish very low levels of fluorescence.
The Auto-Zero feature shutters the photomultiplier tube in order to produce a “dark” scan
with is the cumulative result of all electronic noise and offsets in the system.
The Agilent 3100 Bioanalyzer uses multi-color fluorescence based nucleic acid analysis
system. The technology of capillary electrophoreses with 16 capillaries operating in
parallel to analyze 12 samples simultaneously. The Bioanalyzer is connected to a
computer workstation loaded with 3100 Data Collection software to control, monitor, and
collect data. This instrument is used to assess the quality of RNA and DNA samples
prior to subjecting them to microarray analysis.
The Nanodrop Spectrophotometer uses only 1µl of sample and measures from 220 to 320
nm simultaneously. This gives a good indication of potential chemical contaminant carry
over during processing that can interfere with downstream applications.
The Affymetrix Core Facility is managed by Julia Gouffon who has her Masters in
Microbiology and fifteen years technical experience including 5 years array experience at
UTK and ORNL.