P-CAGE-26288 - P-CAGE-26288
For each hybridization 250 pmol of Cy3 and Cy5 probes were mixed, dried in speed-vac, and resuspended in 9 ul of RNase-free water. Labelled aRNA was fragmented by adding 1 ul of 10x Fragmentation buffer (Ambion) and incubating at 70°C for 15 minutes. The reaction was stopped with 1 ul of Stop solution (Ambion). Integrity and average size of aRNA and fragmented aRNA were evaluated using the Bioanalyzer 2100. The final volume of the probe was diluted to 100 ul in hibridization solution. Printed slides were rehydrated over a 65°C water bath for 10 seconds and dried on a 65°C heating block for 10 seconds. This hydration step was repeated three times. Oligonucleotides were fixed by UV radiation of 120mJ. Slides were washed in 1% SDS for 5 minutes, H2O for 5 minutes and in absolute ethanol for 30 seconds. Finally, slides were dried by centrifugation at 141g for 3 minutes. Prehybridization was performed at 42°C for 30-45 minutes in 6x SSC, 0.5% SDS and 1% BSA. Slides were rinsed five times with distilled water. Cy5 and Cy3 amplified RNA (aRNA) fragmented probes were mixed (250 pmol of each label) with 20 ug of PolyA (Sigma) and 20 ug of yeast tRNA (Sigma) in a final volume of 100 ul of hybridization buffer (50% formamide, 6x SSC, 0.5% SDS, 5x Denhardt's). The probe was denatured at 95°C for 5 minutes and applied to the slide using a LifterSlip (Erie Scientific). Slides were then incubated at 37°C for 16h in hybridization chambers (Array-It). After incubation, slides were washed twice with 0.5x SSC, 0.1% SDS for 5 minutes each, twice with 0.5x SSC for 5 minutes and finally in 0.05x SSC for 5 minutes. Slides were dried by centrifugation at 563g for 1 minute.
Chamber type, Quantity of label target used, Temperature