Total RNA and 2.5 ug oligo dT are brought to 25 ul and denatured for 5 min at 70 C. The reaction is then cooled to 42 C and an equal volume of RT reaction mix, 400 U SuperScript II, modified nucleotides, and 20 U RNasin is added. After appropriate incubation, the reaction is denatured, then neutralized, the buffer removed, and the cDNA dried. Dyes are coupled to the cDNA in the dark at room temperature (1 h). The reaction is then quenched.
Slides are pre-hybridized in 5X-SSC, 0.1% SDS, 1% BSA at 42 C for 45 min. Rinsed 5X in deionized H20 and 1X in isopropanol and dried by centrifugation for 1 min at 1000 rpm. The cDNA hybridization mix was combined with mouse Cot-1 DNA and Oligo dT buffers and incubated overnight at 42 C in a Corning hybridization chamber. Coverslip removed in 2X SSC, 0.1% SDS at room temp. Slide was washed once in 0.2X SSC, 0.1% SDS (42 C, 5 min, with agitation), once in 0.2X SSC (room temperature, 5 min, with agitation) and dried by centrifugation at 1000 rpm for 3 min.
Extracted Cells or dissected tissue were homogenized in Trizol. Following chlorophorm extraction, 1 volume 70% EtOH was added to the aqueous phase and applied to RNeasy column (Qiagen). Extraction followed manufacturer recommendations
Cells were grown in P-100 tissue culture plates. Growth Medium RPMI-1640 was supplemented with 10% fetal calf serum, 10mM Hepes, 2mM L-glutamine, 1mM Sodium-pyruvate, and 0.05 mM 2-mercaptoethanol was changed every 2-3 days. Cells were grown to confluency.