Affymetrix Gene Expression
The DNA Sequencing Facility has available an Affymetrix Gene Expression Instrument catering to in house and local users. Using Affymetrix Gene Expression GeneChips, investigators are able to examine differential gene expression in a wide variety of cell types and tissues. Gene transcripts profiling using Affymetrix' WT arrays is a well-proven technique for expression research and biomarker discovery. There are currently over a dozen types of GeneChips available including Yeast, Human, Rat, Mice. Soon, over 200 microbial species will be available as well as plant types including wheat, corn and poplar. Investigators at the University of Tennessee have used GeneChips to study obesity and lipid metabolism, environmental toxins, and certain cancers.
The Gene Chip 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 genes/EST. Each probe set contains a perfect match and a single base mismatch to serve as a control for non-specific binding.
Array prices are based on hours worked with the client purchasing all necessary supplies. We recommend visiting Affymetrix's web page to discover what projects are available that may help in your research.
The Sequencing facility has the latest Affymetrix Gene Chip Instrumentation including a Gene Chip 640 Hybridization Oven, a Gene chip 450 Fluidics Station and a GeneChip 7G Scanner. The Facility uses an Agilent 2100 Bioanalyzer to assess RNA quality and a Nanodrop Spectrophotometer for the most accurate quantification of small amounts of RNA.
The Gene chip 640 Hybridization oven incubates chips in a rotisserie fashion for thorough hybridization across the entire array. The Gene Chip 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 Gene Chip 7G Scanner uses the Affymetrix patented flying objective lens (FOL) technology with increased scannning speed, decreased optical distortion and excellent emmision path alignment robustness. The resolution of the laser beam is an excitation spot size of 1.5 µm. Gene Chip features are currently set at 11 µm. The Scanner can distinguish very low levels of fluorescence. The Auto-Zero feature shutters the photomultiplier tube inorder to produce a dark scan which is the cumulative result of all electronic noise and offsets in the system.
Perhaps the most important step in microarray analysis is the RNA preparation prior to processing. Great care must be taken to ensure that the RNA is kept from RNAse to prevent degradation. Even if the tissues are snap frozen during the harvest, they must be thawed prior to homogenization and are exposed to RNAse during this critical time. It is recommended that commercially available solutions such as Ambion's RNA later are used for sample storage. Each sample will be run on the Agilent Bioanlayzer prior to starting the process and if the samples show significant degradation they will not be run on arrays. Take extra precaution to prevent RNA degradation.
The first step of processing Affymetrix microarrays is synthesis of cDNA. The standard method requires 1 to 15 µg of total RNA, however the concentration must minimally be 0.5 µg/µl for the first reaction volumes to be correct. There is a small scale method that involves amplification for 200 ng samples. If you cannot acquire the necessary amount, let the Core know and they will order the kit for processing small scale samples.