cbil.upenn.edu:RAD.Protocol:5896:Protocol - cbil.upenn.edu:RAD.Protocol:5896:Protocol

Labeled RNA was dried to 27 ul using a vacuum drier at low heat setting. In order to facilitate hybridization efficiency, labeled RNA was fragmented using a fragmentation reagent (Ambion, Austin, TX). 3 ul of 10X fragmentation reagent were added and the mixture was incubated at 70 C for 15 minutes. The fragmentation reaction was inhibited by the addition of 3 ul stop reagent (Ambion, Austin, TX). Each Cy5-conjugated RNA was combined with an equal volume of Cy3-conjugated RNA. Nuclease-free water was added to each sample to a total volume of 70 ul followed by the addition of 1 ul of 10 mg/ml herring sperm DNA. 71 ul of 2X hybridization buffer (Proteomics Research Solutions Cat. No. PRS-16003050, Ann Arbor, MI) were added to the samples and they were incubated at 95 C for 5 minutes and centrifuged at 10,000g for 1 minute. Samples were loaded onto custom-printed oligo microarrays. Microarrays were hybridized in a Genomics Solution HybStation (Ann Arbor, MI) using a step-down protocol (42 C, 35 C, 30 C each for 5 hours). Microarrays were later washed with medium stringency buffer (PRS-16004001, Proteomics Research Solutions, Ann Arbor, MI) at 30 C for 2 minutes, followed by a high stringency buffer (PRS-16004501, Proteomics Research Solutions, Ann Arbor, MI) wash at 25 C for 2 minutes. Finally, microarrays were washed with post wash buffer (PRS-16003501, Proteomics Research Solutions, Ann Arbor, MI) at 25 C for 2 minutes. They were later dipped into deionized water for 30 seconds and were dried by centrifugation at 500 g for 1 minute. When there were more than 12 assays, hybridizations were done in batches of 12 in random order over several days.
All experiments using protocol cbil.upenn.edu:RAD.Protocol:5896:Protocol: (E-CBIL-42, E-CBIL-43, E-CBIL-47)