Medical College of Wisconsin/Department of Physiology
Manufacture protocol
The custom microarrays were manufactured at the Department of Physiology at the Medical College of wisconsin. The microarrays were printed witha GMS 417 robotic arrayer from Affymetrix (made by Genetic Microsystesm that has a printhead with 4 pin and ring print tips. Arrays were prepared from 70 mer oligonucleotides that correlated to approximately 128 genes related to angiogenesis and approximately 12 genes considered to be positive controls;positive control genes were either genes considered to be generally highly expressed genes in the kidney, and/or genes known to be influenced by ischemia reperfusion injury in the rat kidney. In addition, 10 cDNA derived from Research genetics clone sets were included for comparison but were not included in the final analysis. All oligonucleotides and cDNA were resuspended in 50% DMSO and printed onto poly-L-lysine coated glass slides.Negeative control spots were included thatconsisted of 50% water/50% DMSO. Each gene was printed on each slide between 6-12 times. Blank spots, included for background determination were printed 96 times. Spots were printed with 300 microns center to center, with a spot diameter of approximately 120-150 mucrons. Print configuration is in 12 blocks in a 2 X 6 arrangement and a total of 1152 spots per slide. Positive controls were included in each block. After printing, spoted nucleotides were cross linked to the slides by UV irradiation at 65 mJ (Stratalinker UV cross-linker, Stratagene) Thge microarrays were post processed according to the procedures on the Stanform website (http;// cmgm.stanford.edu/pbrown/protocols/index.html) and subsequently stored at room temperatures in a dessicated chamer until use.
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