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Microarray Technology:
Microarray technology, through nucleic acid hybridization and recent computer advancements, has given us the power to evaluate RNA expression profiles for thousands of genes with a single experiment.
Affymetrix GeneChips® use a patented methodology for building oligonucleotides, in 25-mers, directly onto the glass substrate of the chip. These oligonucleotides are organized in probe pairs of perfect matches and mismatch sequences. The perfect match sequence is exactly complimentary to the specific gene, which is then used to measure the expression. The mismatch sequence differs from the perfect match sequence by a single base at the center (13th) position; this helps to determine the background and nonspecific hybridization that contributes to the signal of the perfect match. There are from 16 to 20 probe matches to one probe set that are scattered over the entire chip. Probes are chosen based on current information from a variety of nucleotide repositories. The software automatically compares the signals of the matching probes and using statistical algorithms gives the probe either a present, absent, or marginal call.
Affymetrix Equipment:
GeneChip Hybridization Oven(not pictured): A "rotisserie" oven is used to hybridize the chip for 16 hours over night at 45°C at 60 rpm. The oven is capable of hybridization of 64 arrays at one time.
Affymetrix GeneChip Fluidics Station 450: The fluidics station contains four modules that hold one GeneChip cartridge each. The modules are controlled independently through the Gene Chip Operating System (GCOS) software. The station performs all the washing and staining of the probe arrays. Chips are stained with Streptavidin-Phycoerythrin and washed with stringent and non-stringent buffer solutions.
Affymetrix GeneChip® Scanner: This scanner contains a solid-state laser to measure fluorescence intensity emitted by cRNA bound to the GeneChip Arrays.
Computer Workstation: The facility has two computer workstations; one station is used to run operating procedures for the fluidics station and the scanner, and the other station is equipped with analysis software. |