title: Brandeis/Harvard Drosophila Melanogaster 3.7K
status: Public on Aug 17 2006
contact.city: Boston
contact.first: Meghana
contact.middle: M
contact.last: Kulkarni
contact.email: mkulkarni@receptor.med.harvard.edu
contact.state: MA
contact.address: 77 Avenue Louis Pasteur
contact.department: Genetics
contact.country: USA
contact.web_link: http://genepath.med.harvard.edu/~perrimon/
contact.institute: Harvard Medical School
organism: Drosophila melanogaster
manufacture_protocol: Custom cDNA-based Microarray: DNA probes to a set of approximately 3,000 strategically selected genes were generated by PCR. These probes were obtained from the PCR amplicons used to generate the dsRNA library 16. The 3,000 genes represented on the array include genes for all the Kinases (350) and Phosphatases (161) encoded in the Drosophila genome, known/canonical components and targets of the major signaling pathways such as Wg/Wnt, Hh, TGF-β, JAK/STAT, NFκB, Notch, RTK, JNK, and INR pathways 33,34; Life cycle website at Yale. Approximately 1,000 of the 3,000 genes were randomly selected to facilitate normalization of signal intensity across the array. Only PCR products showing a clear and strong bands were recovered using Montage PCR plates (Millipore). The DNA concentration was determined using PicoGreen reagent (Molecular Probes). The final concentration of DNA averaged 400 ng/µl. Samples were transferred to 384 well plates (Genetix: x6004) containing DMSO to give a final concentration of 200 ng/µl in 50% DMSO. The DNA probes were directly spotted onto Corning UltraGAPS coated glass slides using a Genetix QArray robot in the Biopolymer Facility at Harvard Medical School. Spots were arrayed in a 14 x 14 arrangement using 24 (4 x 6) Genetix 150µm diameter tip solid microarray pins with a center-to-center spacing of 315 µm. A number of controls were prepared and printed on the glass slides with the probe DNAs. These controls include Negative controls (water, 50% DMSO, sonicated salmon sperm DNA, human Cot1, mouse Cot1, GFP, CFP, YFP, LacZ, and Luciferase) to assess the degree of nonspecific hybridization, and Spiked controls (Lucidea Universal Scorecard, Amersham Biosciences).