O papel modulador do gene AIRE - capes

Complex cDNA probe preparation and hybridizationIn this study, we refer to the radioactive cDNA originated from the thymusRNA samples as the complex probe and the PCR product originated from theclones and deposited on the nylon microarrays as the target.The33 P-labeled cDNA complex probes were prepared by reversetranscription of 10 µg of thymus total RNA using oligo dT 12-18 as a primer. Onehundred microliters of 33 P-cDNA complex probe containing 30- 50-million cpmwas hybridized with nylon microarrays, as previously described [24-27].Imaging acquisitionImaging plates and a phosphor imager storage system (Cyclone model,Packard Instruments, USA) were used to capture the hybridization signals andthe Array Vision ® (Imaging Research Inc. USA) was used to quantify thesignals with local background subtraction, whose spots were matched with atemplate grid.The ratio between vector probe hybridization values and complex probehybridization values for each spot was used as a reference normalization value.Total intensity normalization, using the median expression value, was also used[28].242