CaSki were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration. Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine. Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A. Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs. Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated CaSki was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE). This is a dye-flip experiment to CaSki2v5 (GSM37894).
